Reducing cerebral microvascular amyloid-beta protein deposition diminishes regional neuroinflammation in vasculotropic mutant amyloid precursor protein transgenic mice

Abstract

Cerebral microvascular amyloid-beta (Abeta) protein deposition is emerging as an important contributory factor to neuroinflammation and dementia in Alzheimer's disease and related familial cerebral amyloid angiopathy disorders. In particular, cerebral microvascular amyloid deposition, but not parenchymal amyloid, is more often correlated with dementia. Recently, we generated transgenic mice (Tg-SwDI) expressing the vasculotropic Dutch (E693Q)/Iowa (D694N) mutant human Abeta precursor protein in brain that accumulate abundant cerebral microvascular fibrillar amyloid deposits. In the present study, our aim was to assess how the presence or absence of fibrillar Abeta deposition in the cerebral microvasculature affects neuroinflammation in Tg-SwDI mice. Using Tg-SwDI mice bred onto an apolipoprotein E gene knock-out background, we found a strong reduction of fibrillar cerebral microvascular Abeta deposition, which was accompanied by a sharp decrease in microvascular-associated neuroinflammatory cells and interleukin-1beta levels. Quantitative immunochemical measurements showed that this reduction of the neuroinflammation occurred in the absence of lowering the levels of total Abeta40/Abeta42 or soluble Abeta oligomers in brain. These findings suggest that specifically reducing cerebral microvascular fibrillar Abeta deposition, in the absence of lowering either the total amount of Abeta or soluble Abeta oligomers in brain, may be sufficient to ameliorate microvascular amyloid-associated neuroinflammation.

Figure 1.

Tg-SwDI mice develop fibrillar Aβ deposits exclusively in the cerebral microvasculature. A, Abundant Aβ immunostaining, but lack of thioflavin-S (Th-S) amyloid staining, of diffuse parenchymal deposits in the neocortex of Tg-SwDI mice. B, Colocalization of Aβ immunostaining and thioflavin-S amyloid staining in the thalamus of Tg-SwDI mice is predominantly localized in microvasculature structures. C, Colocalization of vascular collagen IV immunostaining and thioflavin-S amyloid staining in the thalamus of Tg-SwDI mice. Scale bars, 50 μm.

Figure 2.

Lack of endogenous mouse apoE lowers cerebral Aβ deposition in Tg-SwDI mice. A-D, Deposition of Aβ in forebrain (A, B) and frontotemporal cortex (C, D) in Tg-SwDI in the absence or presence, respectively, of endogenous mouse apoE. Scale bars: A, B, 1 mm; C, D, 50 μm, respectively. E, Quantitative image analysis of regional Aβ deposition in Tg-SwDI mice in the presence (black bars) or absence (gray bars) of endogenous mouse apoE. Data shown are mean ± SD (n=10). ^*p < 0.01; ^**p < 0.02; ^#p < 0.001. F/T, Frontotemporal; KO, knock-out.

Figure 3.

Lack of endogenous mouse apoE eliminates cerebral microvascular Aβ deposition in Tg-SwDI mice. A-D, Fibrillar microvascular amyloid deposition in Tg-SwDI mouse thalamus revealed by immunostaining for Aβ (brown) and collagen type IV (red; A, B) and thioflavin-S fluorescence staining (C, D) in the presence (A, C) or absence (B, D) of endogenous mouse apoE. Scale bars, 50 μm. E, Quantitative stereological estimation of microvascular amyloid deposition in the presence (black bars) or absence (gray bars) of endogenous apoE. Data shown are mean ± SD (n = 10). ^*p < 0.01; ^**p < 0.0001. F/T, Frontotemporal; KO, knock-out.

Figure 4.

Effects of endogenous mouse apoE on cerebral human AβPP and Aβ levels in Tg-SwDI mice. A, Immunoblot analysis of transgene-encoded human AβPP levels in mouse forebrain extracts. B, Quantitation of human AβPP immunoblots (n = 4) of mouse forebrain extracts. C, ELISA measurements of total Aβ40 (black bars) and Aβ42 (gray bars) in mouse forebrain tissue. ELISA data shown are mean ± SD (n = 12-16). a.u., Arbitrary units; KO, knock-out.

Figure 5.

Effects of endogenous mouse apoE on cerebral soluble and insoluble Aβ levels in Tg-SwDI mice. A, Ratio of total insoluble Aβ/soluble Aβ. B, Representative dot-blot analysis of Aβ oligomers in soluble mouse forebrain extracts. C, Quantitation of soluble Aβ oligomers from dot blots of Tg-SwDI and Tg-SwDI/apoE^-/- mice. Data shown are mean ± SD (n = 5). ^*p < 0.001. KO, Knock-out.

Figure 6.

Reduced cerebral microvascular amyloid decreases reactive astrocytes in Tg-SwDI mice. A-D, Microvascular-associated reactive astrocytes revealed by GFAP-positive immunostaining (brown) and collagen type IV (red) in Tg-SwDI mouse frontotemporal cortex (A, B) or thalamus (C, D) in the presence (A, C) or absence (B, D) of endogenous mouse apoE. Scale bars, 50 μm. E, Quantitative stereological estimation of reactive astrocyte densities in brain regions of Tg-SwDI mice in the presence (black bars) or absence (gray bars) of endogenous mouse apoE. Data shown are mean ± SD (n = 10). ^*p < 0.001. F/T, Frontotemporal; KO, knock-out.

Figure 7.

Reduced cerebral microvascular amyloid decreases activated microglia in Tg-SwDI mice. A-D, Microvascular-associated activated microglia revealed by 5D4-positive immunostaining (brown) and collagen type IV (red) in Tg-SwDI mouse frontotemporal cortex (A, B) or thalamus (C, D) in the presence (A, C) or absence (B, D) of endogenous mouse apoE. Scale bars, 50 μm. E, Quantitative stereological estimation of activated microglial densities in brain regions of Tg-SwDI mice in the presence (black bars) or absence (gray bars) of endogenous mouse apoE. Data shown are mean ± SD (n = 10). ^*p < 0.0001; ^**p < 0.01. F/T, Frontotemporal; KO, knock-out.

Figure 8.

Reduced cerebral microvascular amyloid lowers the elevated IL-1β levels found in Tg-SwDI mice. The levels of IL-1β were measured in soluble forebrain extracts of 12-month-old wild-type, Tg-SwDI/apoE^+/+, and Tg-SwDI/apoE^-/- mice by ELISA analysis. Data shown are mean ± SD (n = 5). ^*p < 0.0002; ^**p < 0.01. KO, Knock-out.