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. 2011 Dec;70(12):1124-37.
doi: 10.1097/NEN.0b013e31823b0b12.

Changes in brain β-amyloid deposition and aquaporin 4 levels in response to altered agrin expression in mice

Steven M Rauch  1 , Kathy Huen, Miles C Miller, Hira Chaudry, Melissa Lau, Joshua R Sanes, Conrad E Johanson, Edward G Stopa, Robert W Burgess
Affiliations

Changes in brain β-amyloid deposition and aquaporin 4 levels in response to altered agrin expression in mice

Steven M Rauch et al. J Neuropathol Exp Neurol. 2011 Dec.

Abstract

Conditions that compromise the blood-brain barrier (BBB) have been increasingly implicated in the pathogenesis of Alzheimer disease (AD). AGRIN is a heparan sulfate proteoglycan found abundantly in basement membranes of the cerebral vasculature, where it has been proposed to serve a functional role in the BBB. Furthermore, AGRIN is the major heparan sulfate proteoglycan associated with amyloid plaques in AD brains. To examine the relationship of AGRIN, the BBB, and AD-related pathologies, we generated mice in which the Agrn gene was deleted from either endothelial cells or neurons using gene targeting or was overexpressed using a genomic transgene construct. These mice were combined with a transgenic model of AD that over expresses disease-associated forms of amyloid precursor protein and presenilin 1. In mice lacking endothelial cell expression of Agrn, the BBB remained intact but aquaporin 4 levels were reduced, indicating that the loss of AGRIN affects BBB-associated components. This change in Agrn resulted in an increase in β-amyloid (Aβ) in the brain. Conversely, overexpression of Agrn decreased Aβ deposition, whereas elimination of Agrn from neurons did not change Aβ levels. These results indicate that AGRIN is important for maintaining BBB composition and that changes in Agrn expression (particularly vessel-associated AGRIN) influence Aβ homeostasis in mouse models of AD.

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Figures

Figure 1
Figure 1
AGRIN deletion from endothelial cells. Combining the Agrinfl/fl allele with transgenic Tie2-Cre expression results in a reduction in blood vessel-associated AGRIN in the brain, as detected by immunostaining in adult mice (6 months of age and greater). (A) By indirect immunofluorescence, cerebral blood vessels and the pia separating the cortex and midbrain in a control mouse are strongly immunoreactive for carboxy-terminal anti-AGRIN. (B) The anti-AGRIN staining intensity is reduced to near the threshold of detection in Agrnfl/fl; Tie2-Cre mice, although the pia remains intensely labeled. (C, D) Using more sensitive enhanced peroxidase immunohistochemistry and a long isoform (LN-AGRIN)-specific amino-terminal antibody, anti-AGRIN labeling is reduced but not eliminated from the cerebral vasculature in Agrnfl/fl; Tie2-Cre samples. (E, F) Vessel morphology and the reduction in staining intensity at higher magnification.
Figure 2
Figure 2
Effects of endothelial cell Agrn deletion on blood-brain barrier composition. (A, B) FITC dextran and sulforhodamine injected intravenously did not enter the brain parenchyma in either control (A) or Agrnfl/fl; Tie2-Cre (B) samples from mice ages 6 months and greater. Arrows denote blood vessels. (C, D) Transmission electron microscopy of cerebral basement membrane on the abluminal surface of endothelial cells (between arrows) in control (C) and Agrnfl/fl; Tie2-Cre (D) mice does not reveal any thinning or breaches in the structural integrity of the matrix. (E) Elemental spectroscopy analysis of the basement membrane revealed a decrease in the ratio of sulfur to phosphorus following endothelial cell Agrn deletion, suggesting that the decrease in AGRIN is not compensated for by an increase in other sulfated proteoglycans. Results are from 8 independent fields examined in each of 2 mice of each genotype. (F, G) Cerebral blood vessels labeled with anti-AGRIN in control (F) and Agrnfl/fl; Tie2-Cre (G) mice demonstrate a reduction in labeling surrounding the vessels, smaller vessel diameters, and more ragged profiles in the Agrnfl/fl; Tie2-Cre samples, consistent with changes in the neurovascular unit in response to endothelial cell Agrn deletion. (H–K) Immunostaining for Aquaporin4 (AQP4) in wild type (H, J) and Agrnfl/fl; Tie2-Cre (I, K) mice demonstrates a marked decrease in immunoreactivity following endothelial cell Agrn deletion. Higher magnification panels (J, K) indicate that the localization of the AQP4 was not changed. (L) An enzyme-linked immunosorbent assay (ELISA) was used to quantify AQP4 in brain homogenates from age and strain matched control mice (n = 6), Agrnfl/fl mice with no transgenic Cre expression (n = 3), and Agrnfl/fl; Tie2-Cre mice lacking endothelial cell Agrin expression (n = 6). Consistent with the reduced staining intensity in immunocytochemistry, AQP4 levels were reduced from 0.68 ± 0.15 ng AQP4/mg of tissue in wild type controls vs. 0.28 ± 0.07 in Agrnfl/fl; Tie2-Cre mice (mean ± SD, t test. p = 0.0002).
Figure 3
Figure 3
Deletion of short isoform (SN)-Agrn. (A) The single exon encoding the SN amino terminus of AGRIN and approximately 2 kb of upstream sequence were targeted by homologous recombination; upstream exons encoding the long isoform (LN)-N-terminus and downstream exons encoding common sequences were left intact. The loxP-flanked selectable marker, Neo, for embryonic stem cells was removed from the genome by mating to Cre transgenic mice and a single loxP site and flanking restriction sites were left in the Agrn gene. Southern blotting DNA samples from control and homozygous SN-deletion mice confirmed the homologous recombination event with the anticipated shifts in restriction fragment sizes. (B) In situ hybridization with a probe specific to the SN-isoform of Agrn revealed strong expression in the adult hippocampus. (C) Hybridization with a probe recognizing all Agrn isoforms showed a similar pattern of expression. D, E: Hybridization with SN-specific (D) or pan-Agrn (E) probes indicated a lack of Agrn expression in the hippocampus of adult SN-Agrn knockout mice, indicating that SN-Agrn is the predominant isoform expressed in the hippocampus. (F, G) Adult neuromuscular junctions stained with α-bungarotoxin to label AChRs (red) and an antibody against the C-terminus of AGRIN (green) revealed no changed in AGRIN localization or NMJ morphology in the SN-Agrn knockout mice. (H, I) Kidney glomeruli stained with an antibody against the C-terminus of AGRIN revealed abundant basement membrane staining in both control (H) and SN-Agrn knockout (I) kidneys. Data in panels F–I are consistent with preserved expression of LN- AGRIN. (J) SN-AGRIN is the predominant isoform in brain. In AChR clustering activity assays, homogenates from SN-Agrn knockout brains were reduced in clustering activity by 4- to 5-fold compared to homogenates from control brains (n = 3 mice of each genotype), indicating SN-AGRIN is the predominant Z+ isoform of AGRIN expressed by CNS neurons. Background AChR clustering activity was determined by treating myotube cultures with recombinant inactive AGRIN and 100% activity was determined using saturating doses of recombinant Z8 AGRIN.
Figure 4
Figure 4
Composition of APP/PS1 transgenic mouse plaques. Plaques are detectable in the brains of APP/PS1 mice beginning between 5 and 6 months of age. (A) Low magnification of plaques stained with 4G8 (red), a monoclonal antibody specific for human β-Amyloid (Aβ); nuclei are counterstained in blue (DAPI). (B) High magnification of a plaque stained with 4G8 shows a dense core of Aβ surrounded by fibrillar protein. (C) Plaques are also reactive for acetylcholine esterase (AChE, brown). (D) Activated astrocytes detected with anti-glial fibrillary acidic protein (GFAP, green) surround plaques labeled with 4G8 (red). (E) Plaques in the transgenic mice are also positive for heparan sulfate proteoglycans (HSPG, green). (F) Filter trap analysis of SDS-insoluble Aβ. Nine mice were examined at 6 most of age; they consistent of 3 male APP/PS1 transgenics (columns 1–3), a non-transgenic wild type mouse (column 4), and 5 female APP/PS1 transgenics (columns 5–9). Each column represents a dilution series for each sample, amounts of total protein loaded per well are 100 μg in the top row, 80, 60, 40 and 20 μg in the bottom row. Note the consistency of the technique and the relative quantification (20–40 μg of protein in females is equivalent in intensity to 80 to 100 μg of protein in males, consistent with other results indicating that females express approximately 3-fold higher levels of Aβ). The male/female difference has been reported by others and provided an internal control for subsequent assays. Note the total lack of signal in column 4, the non-transgenic mouse.
Figure 5
Figure 5
β-Amyloid (Aβ) levels in mice lacking endothelial cell or short isoform (SN)-Agrn. (A) Female mice 6 months of age and lacking endothelial cell AGRIN (Agrnfl/fl; Tie2-Cre) showed significantly higher levels of Aβ-40 vs. control mice (t test p value = 0.018); levels of Aβ-42 and total Aβ were not different. Resul ts are reported as mean percent of control values ± SEM; animal numbers are indicated. (B) Aβ in SN-Agrn KO mice. Deletion of SN-Agrn did not result in significant differences from controls in Aβ levels at 6 months of age, as determined by ELISA analysis. Data shown are for male mice; values are mean percent of control ± SEM; animal numbers are indicated.
Figure 6
Figure 6
Effect of transgenic Agrn overexpression on β-Amyloid (Aβ) levels. (A) Staining with an anti-GFP antibody detects transgenic AGRIN-CFP in both plaques and blood vessels, indicating that the transgenic protein localizes to plaques and the blood-brain barrier. Note the staining intensity is much higher in the blood vessel (arrow) than in the plaque. (B) In filter trap assays, 6-month-old Agrn-CFP transgenic mice had significantly reduced SDS-insoluble Aβ levels vs. age-matched control littermates. Data shown are for male mice only; mean relative signal intensity based on densitometry ± SD, t test p = 0.02; animal numbers are shown. (C) Levels of Aβ were further examined by ELISA in 6-month-old mice. Transgenic expression of Agrn-CFP significantly reduced total amyloid, Aβ-42, and Aβ-40 levels compared to littermate controls. Asterisks indicate t test p values <0.05, animal numbers are given. Results shown are for male mice, values are mean percent of control values ± SEM. (D) Further subdividing Aβ species into DEA-soluble and insoluble fractions, indicated that the greatest contribution of the reduction seen with Agrn-CFP overexpression came from reduced Aβ-42 insoluble amyloid, although differences did not reach statistical significance (p values 0.06–0.08).
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