Early-onset subicular microvascular amyloid and neuroinflammation correlate with behavioral deficits in vasculotropic mutant amyloid beta-protein precursor transgenic mice

Abstract

Cerebral microvascular amyloid beta protein (Abeta) deposition and associated neuroinflammation are increasingly recognized as an important component leading to cognitive impairment in Alzheimer's disease and related cerebral amyloid angiopathy (CAA) disorders. Transgenic mice expressing the vasculotropic Dutch/Iowa (E693Q/D694N) mutant human Abeta precursor protein in brain (Tg-SwDI) accumulate abundant cerebral microvascular fibrillar amyloid deposits exhibiting robust neuroinflammation. In the present study, we sought to determine if the unique amyloid pathology of Tg-SwDI mice was associated with deficits in behavioral performance. Behavioral performance tests that assessed a variety of psychological functions, including overall activity, motor ability, balance and strength, anxiety, impulsivity, and learning were conducted on homozygous Tg-SwDI mice and similarly aged wild-type C57Bl/6 mice. Our results indicate that Tg-SwDI mice were impaired in the performance of the Barnes maze learning and memory task at 3, 9, and 12 months of age. While more widespread cerebral microvascular Abeta pathology was evident in older animals, the evaluation of the Abeta pathology in the 3 months old transgenic animals revealed specific accumulation of microvascular amyloid and markedly elevated numbers of reactive astrocytes and activated microglia restricted to the subiculum. These findings indicate that early-onset accumulation of subicular microvascular amyloid and accompanying neuroinflammation correlates with impaired performance in the learning and memory task in Tg-SwDI mice.

Figure 1

Homozygous Tg-SwDI mice accumulate markedly more Aβ than heterozygous animals. Aβ immunostaining in twelve months old heterozygous (A) homozygous (B) Tg-SwDI reveals more extensive deposition in the latter. Scale bar = 1 mm. (C) Total Aβ 40 and Aβ 42 loads in homozygous Tg-SwDI mice is more than double compared to heterozygous animals. *P < 0.001. (D) The levels of soluble Aβ and insoluble Aβ are similarly increased in homozygous Tg-SwDI mice compared with heterozygous animals. Data presented are the mean ± S.D. (n = 10 per group). *P < 0.0005.

Figure 2

Twelve months old (A) and three months old (B) Tg-SwDI mice exhibited slower rates of decrease in the latencies to find the escape hole in the Barnes Maze spatial learning and memory test compared with age-matched wild type mice. Shown are the best fitting exponential decay curves for each group (12 months old WT y = 93.8^e−.09; 12 months old Tg-SwDI y = 94.1^e−.03; 3 months old WT y = 77.4^e−.15; 3 months old Tg-SwDI y = 88.8^e−.06).

Figure 3

Accumulation of cerebral Aβ in young and older Tg-SwDI mice. (A) ELISA measurements of total Aβ 40 (gray bars) and Aβ 42 (black bars) in mouse forebrain tissue. (B) ELISA measurements of soluble Aβ (gray bars) and insoluble Aβ (black bars) in mouse forebrain tissue. ELISA data shown are mean ± S.D. (n = 6 mice per age group). *P < 0.000001, **P < 0.0001. (C) Representative dot blot analysis of Aβ oligomers in soluble mouse forebrain extracts. (D) Quantitation of soluble Aβ oligomers from dot blots of young and older Tg-SwDI mice. Data shown are mean ± S.D. (n = 6 mice per age group). *P < 0.0002.

Figure 4

Cerebral microvascular amyloid accumulation in young and older Tg-SwDI mice. Microvascular amyloid deposition in Tg-SwDI mouse brain revealed by immunostaining for Aβ (brown) and collagen type IV for identification of microvessels (red) in the fronto-temporal cortex (A,E), thalamus (B,F), and subiculum (C,G) from twelve months old and three months old Tg-SwDI mice, respectively. Scale bars = 50 μm. Quantitative stereological measurement of cerebral microvascular Aβ deposition in different brain regions of twelve months old (D) and three months old (H) Tg-SwDI mice. Data shown are the mean ± S.D. (n = 6 mice per age group).

Figure 5

Elevated numbers of reactive astrocytes in the subiculum of young Tg-SwDI mice. Microvascular-associated reactive astrocytes revealed by GFAP-positive immunostaining (brown) and collagen type IV for identification of microvessels (red) in the fronto-temporal cortex (A,E), thalamus (B,F), and subiculum (C,G) from twelve months old and three months old Tg-SwDI mice, respectively. Scale bars = 50 μm. Quantitative stereological measurement of reactive astrocyte densities in different brain regions of twelve months old (D) and three months old (H) wild-type (gray bars) or Tg-SwDI mice (black bars). Data shown are the mean ± S.D. (n = 6 mice per age group).

Figure 6

Elevated numbers of activated microglia in the subiculum of young Tg-SwDI mice. Microvascular-associated activated microglia revealed by 5D4-positive immunostaining (brown) and collagen type IV for identification of microvessels (red) in the fronto-temporal cortex (A,E), thalamus (B,F), and subiculum (C,G) from twelve months old and three months old Tg-SwDI mice, respectively. Scale bars = 50 μm. Quantitative stereological measurement of activated microglia densities in different brain regions of twelve months old (D) and three months old (H) wild-type (gray bars) or Tg-SwDI mice (black bars). Data shown are the mean ± S.D. (n = 6 mice per age group).