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Comparative Study
. 2009 Feb 11;29(6):1846-54.
doi: 10.1523/JNEUROSCI.5715-08.2009.

Induction of toll-like receptor 9 signaling as a method for ameliorating Alzheimer's disease-related pathology

Henrieta Scholtzova  1 , Richard J Kascsak, Kristyn A Bates, Allal Boutajangout, Daniel J Kerr, Harry C Meeker, Pankaj D Mehta, Daryl S Spinner, Thomas Wisniewski
Affiliations
Comparative Study

Induction of toll-like receptor 9 signaling as a method for ameliorating Alzheimer's disease-related pathology

Henrieta Scholtzova et al. J Neurosci. .

Abstract

The pathogenesis of Alzheimer's disease (AD) is thought to be related to the accumulation of amyloid beta (Abeta) in amyloid deposits and toxic oligomeric species. Immunomodulation is emerging as an effective means of shifting the equilibrium from Abeta accumulation to clearance; however, excessive cell mediated inflammation and cerebral microhemorrhages are two forms of toxicity which can occur with this approach. Vaccination studies have so far mainly targeted the adaptive immune system. In the present study, we have stimulated the innate immune system via the Toll-like receptor 9 (TLR9) with cytosine-guanosine-containing DNA oligodeoxynucleotides in Tg2576 AD model transgenic mice. This treatment produced a 66% and 80% reduction in the cortical (p = 0.0001) and vascular (p = 0.0039) amyloid burden, respectively, compared with nontreated AD mice. This was in association with significant reductions in Abeta42, Abeta40, and Abeta oligomer levels. We also show that treated Tg mice performed similarly to wild-type mice on a radial arm maze. Our data suggest that stimulation of innate immunity via TLR9 is highly effective at reducing the parenchymal and vascular amyloid burden, along with Abeta oligomers, without apparent toxicity.

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Figures

Figure 1.
Figure 1.
Locomotor activity. A–D, At 16 months of age (posttreatment), both Tg groups and their wild-type (Wt) littermates did not differ in any of the locomotor parameters measured: maximum speed (A), distance traveled (B) average speed (C) and resting time (D). The error bars show the SEM. This applies also to all subsequent figures.
Figure 2.
Figure 2.
Working memory improvement in CpG ODN-treated Tg2576 mice. Tg2576 mice treated with CpG ODN navigated the radial arm maze with significantly fewer errors than control Tg mice, and their performance was similar to that of their Wt age-matched littermates [two-way repeated-measures ANOVA, group (treatment) effect, p = 0.019; days effect, p < 0.0001; interaction (group vs days), p = 0.144, Newman–Keuls multiple comparison post hoc testing showed Tg-CpG vs Tg-vehicle, p = 0.026; Tg- vehicle vs Wt, p = 0.039; Tg-CpG vs Wt, p = 0.814].
Figure 3.
Figure 3.
Treatment with CpG ODN decreased cortical and hippocampal amyloid plaque burden in APP Tg2576 mice. A, B, F, G, Histological analysis of APP Tg mice depict the difference in Aβ burden. Aβ immunostaining (6E10/4G8) showed greater Aβ accumulation in cortical (A) and hippocampal (F) sections of vehicle-treated APP mice, compared with sections from CpG ODN- treated APP mice (B, G). C, D, H, I, Similarly, Thioflavin-S cortical and hippocampal staining also revealed differences between vehicle-treated (C, H) and CpG ODN-treated (D, I) APP mice. Stereological analysis of total amyloid burden (Aβ load) showed a significant reduction in APP-Tg mice treated with CpG ODN compared with age-matched Tg control mice treated with vehicle. E, J, There was a 66% reduction in cortical (E) amyloid burden (***p = 0.0001) and a 59% reduction in hippocampal (J) amyloid burden (**p = 0.002) as quantified using unbiased random sampling scheme and semiautomated image analysis system. The scale bar in B corresponds to cortical images A-D. The scale bar in G corresponds to hippocampal images F-I.
Figure 4.
Figure 4.
Aβ burden in the vasculature (CAA burden) and brain microhemorrhages. A–C, Thioflavin-S staining (A, B) revealed a visible reduction in the CAA burden of the penetrating cortical vessels (white arrowhead). There was an 80% decrease (C) in the burden of CAA in CpG ODN-treated Tg2576 mice (Tg-CpG vs Tg-vehicle, **p = 0.0039). D, Quantification of CAA-associated microhemorrhages (Perl's stain) also revealed a significant reduction of iron positive profiles per brain section in CpG-treated group (Tg-CpG vs Tg-vehicle, *p = 0.029).
Figure 5.
Figure 5.
Aβ levels in brain. A, B, Treatment with CpG ODN significantly decreased total (A) and soluble (B) brain Aβ levels in Tg2576 mice. A, Aβ40, 59% reduction, *p = 0.019; Aβ42, 56% reduction,*p = 0.026. B, Aβ40, 75% reduction, **p = 0.003; Aβ42, 74% reduction, **p = 0.0019.
Figure 6.
Figure 6.
Western blot detection and densitometric analysis of A11 immunoreactive oligomer-specific bands. A, B, Western blot (A) of brain homogenates stained with A11 oligomer-specific polyclonal antibody and densitometric analysis (B) of oligomer-specific (56 kDa) band showed significant difference between CpG ODN-treated and vehicle-treated Tg animals (*p = 0.033).
Figure 7.
Figure 7.
CpG ODNs reduced overall cortical and hippocampal CD11b immunoreactivity in APP Tg2576 mice. A–C, Immunostaining (A, B) with CD11b microglial marker followed by semiquantitative analysis (C) revealed significant reduction in cortical and hippocampal (data not shown) microgliosis in CpG ODN-treated (A) compared with vehicle-treated (B) Tg animals (Tg-CpG vs Tg-vehicle, ***p = 0.0001). The degree of microgliosis was graded on a scale from 0 to 3.
Figure 8.
Figure 8.
Reduction in cortical and hippocampal CD45 immunoreactivity (CD45 load) in CpG ODN-treated Tg2576 mice. Cortical (A, B) and hippocampal (D, E) CD45 immunohistochemistry also indicated an overall reduction in microglial activity in CpG ODN- treated mice. Quantitative stereological analysis within the cortex revealed a 71% reduction (***p < 0.001) in CD45 immunoreactivity in CpG ODN-treated Tg mice compared with control Tg mice (C). Likewise, CD45 immunoreactivity within the hippocampus was reduced by 73% (***p < 0.001) in Tg-CpG group compared with Tg-vehicle group (F). The scale bars in B and E correspond to cortical and hippocampal images, respectively.
Figure 9.
Figure 9.
Microglial reactivity around the plaques. A–E, Immunostaining of CD45 (A, B) double-stained with Thioflavin-S (C, D) followed by semiquantitative analysis (E) demonstrated an increase in CD45 immunoreactivity around remaining plaques in the CpG-treated group (Tg-CpG vs Tg-vehicle, *p = 0.047). Representative sections are shown in A–D. Scale bar, 50 μm.
Figure 10.
Figure 10.
Treatment with CpG ODNs reduced cortical GFAP reactive astrocytosis in APP Tg2576 mice. A–D, GFAP immunostaining (A–C) followed by semiquantitative analysis (D) revealed fewer activated astrocytes in CpG ODN-treated Tg animals compared with vehicle-treated animals (Tg-CpG vs Tg-vehicle, **p = 0.006; Tg-vehicle vs Wt, ***p = 0.0005; Tg-CpG vs Wt, p < 0.054). Reactive astrocytosis was rated on a scale of 0.5–3.
Figure 11.
Figure 11.
Levels of autoantibodies. A, B, At 17 months of age there was a significantly higher autoantibody response toward Aβ40 (A) and a trend for a higher response to Aβ42 (B) in CpG ODN-treated Tg mice when compared with vehicle-treated Tg mice. The Tg-vehicle mice did not differ from the wild-type controls. A, Tg-CpG versus Tg-vehicle, *p = 0.017; Tg- vehicle versus Wt, p = 0.24; Tg-CpG versus Wt, *p = 0.042. B, Tg-CpG versus Tg-vehicle, p = 0.09; Tg- vehicle versus Wt, p = 0.44; Tg-CpG versus Wt, p = 0.15. No apparent differences were observed between the groups in 12-month-old animals (data not shown).
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