Amyloid β Oligomers Disrupt Blood-CSF Barrier Integrity by Activating Matrix Metalloproteinases

Abstract

The blood-CSF barrier (BCSFB) consists of a monolayer of choroid plexus epithelial (CPE) cells that maintain CNS homeostasis by producing CSF and restricting the passage of undesirable molecules and pathogens into the brain. Alzheimer's disease is the most common progressive neurodegenerative disorder and is characterized by the presence of amyloid β (Aβ) plaques and neurofibrillary tangles in the brain. Recent research shows that Alzheimer's disease is associated with morphological changes in CPE cells and compromised production of CSF. Here, we studied the direct effects of Aβ on the functionality of the BCSFB. Intracerebroventricular injection of Aβ1-42 oligomers into the cerebral ventricles of mice, a validated Alzheimer's disease model, caused induction of a cascade of detrimental events, including increased inflammatory gene expression in CPE cells and increased levels of proinflammatory cytokines and chemokines in the CSF. It also rapidly affected CPE cell morphology and tight junction protein levels. These changes were associated with loss of BCSFB integrity, as shown by an increase in BCSFB leakage. Aβ1-42 oligomers also increased matrix metalloproteinase (MMP) gene expression in the CPE and its activity in CSF. Interestingly, BCSFB disruption induced by Aβ1-42 oligomers did not occur in the presence of a broad-spectrum MMP inhibitor or in MMP3-deficient mice. These data provide evidence that MMPs are essential for the BCSFB leakage induced by Aβ1-42 oligomers. Our results reveal that Alzheimer's disease-associated soluble Aβ1-42 oligomers induce BCSFB dysfunction and suggest MMPs as a possible therapeutic target.

Significance statement: No treatments are yet available to cure Alzheimer's disease; however, soluble Aβ oligomers are believed to play a crucial role in the neuroinflammation that is observed in this disease. Here, we studied the effect of Aβ oligomers on the often neglected barrier between blood and brain, called the blood-CSF barrier (BCSFB). This BCSFB is formed by the choroid plexus epithelial cells and is important in maintaining brain homeostasis. We observed Aβ oligomer-induced changes in morphology and loss of BCSFB integrity that might play a role in Alzheimer's disease progression. Strikingly, both inhibition of matrix metalloproteinase (MMP) activity and MMP3 deficiency could protect against the detrimental effects of Aβ oligomer. Clearly, our results suggest that MMP inhibition might have therapeutic potential.

Keywords: Alzheimer's disease; amyloid β toxicity; blood–CSF barrier; choroid plexus; matrix metalloproteinases.

Figure 1.

Analysis of inflammation in the hippocampus after intracerebroventricular injection of oligomerized Aβ1–42 in the cerebral ventricles. a–c, mRNA expression analysis of Il1β (a), Il6 (b), and Tnf (c) in the hippocampus 6 h after intracerebroventricular injection of Aβ1–42 oligomers in C57BL/6 mice compared with control hippocampus samples (n = 4). d, e, Representative images of Iba1 staining of brain sections from C57BL/6 mice 6 h after intracerebroventricular injection with scrambled peptide (d) or Aβ1–42 oligomers (e). As indicated on the images, the region surrounding the lateral ventricle is represented. f, Quantification of the percentage of activated microglia in cortex and hippocampus of scrambled and Aβ1–42 oligomer injected mice (n = 5). Scale bar, 25 μm.

Figure 2.

Cytokine and chemokine analyses of CP, hippocampus, and CSF after intracerebroventricular injection of Aβ1–42 oligomers. a–d, mRNA expression analysis of Il1β (a), Il6 (b), Tnf (c), and Inos (d) in CP after intracerebroventricular injection of Aβ1–42 oligomers (gray) in C57BL/6mice compared with control CP samples (black) (n = 3–4). e–h, mRNA expression analysis of Il1β (e), Il6 (f), Tnf (g), and Inos (h), in the hippocampus after intracerebroventricular injection of Aβ1–42 oligomers (gray) in C57BL/6mice compared with control samples (black) (n = 3–4). i–p, Levels of cytokines (IL-1β, IL-6, TNFα, and IFNγ) and chemokines (MIP-1α, MIP-1β, MCP-1, and GM-CSF) in CSF isolated from C57BL/6 mice injected intracerebroventricularly with scrambled peptide (black) or Aβ1–42 oligomers (gray) (n = 4).

Figure 3.

CP morphology analysis by SFB-SEM. a, b, Representative SBF-SEM images of CP cells of C57BL/6 mice injected intracerebroventricularly with scrambled peptide (a) or Aβ1–42 oligomers (b). Cell shape is outlined in green. c, d, 3D modeling (blue) based on merging ∼200 sections of CPE cells from scrambled (c) and Aβ1–42 oligomer (d) injected mice. Only cell shape was considered; basolateral labyrinth and microvilli were neglected while generating the 3D modeling. Mv, Microvilli; Nu, nucleus. Scale bar, 2 μm.

Figure 4.

Analysis of Aβ1–42 oligomer-induced disruption of BCSFB integrity. a, Relative BCSFB permeability 2 and 6 h after intracerebroventricular injection of Aβ1–42 oligomers in the cerebral ventricles (gray) compared with control mice (black) (n = 4). b, c, Ocln (b) and Cldn5 gene (c) expression in CP tissue of C57BL/6 mice injected intracerebroventricularly with scrambled peptide (black) or Aβ1–42 oligomers (gray) (n = 4). d, Western blot analysis of Occludin in CP tissue from C57BL/6 control mice and mice injected intracerebroventricularly with Aβ1–42 oligomers. e–i, Zo1 (e), Cldn1 (f), Zo3 (g), Ecdh (h), and Ncdh (i) gene expression in CP tissue of C57BL/6 mice injected in the cerebral ventricles with Aβ1–42 oligomers (gray) or scrambled peptide (black) (n = 4). j–m, Representative confocal images of C57BL/6 control mice (j, k) and mice injected intracerebroventricularly with Aβ1–42 (l, m) oligomers for 6 h (red, Occludin; blue, Hoechst). The arrowheads point to the apically located tight junctions. Scale bars: e, g, 50 μm; f, h, 10 μm. n = 5. n, o, Representative confocal images of control (n) and Aβ1–42 (o) oligomers injected intracerebroventricularly in C57BL/6 mice stained for E-cadherin (ECDH; red) and Hoechst (blue). The arrowheads point to adherens junctions.

Figure 5.

Analysis of the effect of Aβ1–42 oligomers on BBB integrity. a, Relative permeability of the BBB 6 h after intracerebroventricular injection of Aβ1–42 oligomers (gray) compared with scrambled peptide injected mice (black) (n = 10–13). b–e, Relative gene expression of tight junction proteins Occludin (b), Claudin-5 (c), Zona occludens-1 (d), and Claudin-1 (e) in the hippocampus 6 h after intracerebroventricular injection of scrambled peptide (black) or Aβ1–42 oligomers (gray) (n = 5).

Figure 6.

Analysis of the role of MMPs in Aβ1–42 oligomer-induced disruption of BCSFB permeability. a, Fold change in Mmp gene expression in the CP 2 h (black) and 6 h (gray) after intracerebroventricular injection of Aβ1–42 oligomers in the cerebral ventricles compared with control samples (n = 3–4). b, Total MMP activity in CSF of C57BL/6 mice 6 h after intracerebroventricular injection of scrambled peptide (control), Aβ1–42 oligomers (Aβ), or Aβ1–42 oligomers together with GM6001 (Aβ + GM6001) (n = 6). c, BCSFB permeability of C57BL/6 mice 6 h after intracerebroventricular injection of Aβ1–42 oligomers alone (Aβ) or combined with the MMP inhibitor GM6001 (Aβ + GM6001) compared with scrambled peptide injected mice (control) (n = 6–7). d, Western blot analysis of MMP3 protein levels in CSF of control and Aβ1–42 oligomer intracerebroventricularly injected mice. e, Relative BCSFB permeability in wild-type (black) and MMP3^−/− (gray) C57BL/6 mice 6 h after intracerebroventricular injection of scrambled control or Aβ1–42 oligomers (n = 4–10).

Figure 7.

CP morphology analysis by SFB-SEM of MMP3^−/− mice. a, b, Representative SFB-SEM images of the CPE cells of scrambled peptide (a) and Aβ1–42 oligomer (b) intracerebroventricularly injected MMP3^−/− mice. Cell shape is represented in green. Basolateral labyrinth and microvilli were neglected while drawing the cell outline. Mv, Microvilli; Nu, nucleus.

Figure 8.

Schematic overview of direct effects of Aβ1–42 oligomers on the BCSFB. A monolayer of CPE cells, tightly connected by tight junctions, restricts entrance of molecules from the fenestrated capillaries into the CSF. Injection of oligomerized Aβ1–42 into the cerebral ventricles of mice (1) leads to secretion of MMPs, cytokines, and chemokines from CPE cells into the CSF (2), and this induces disruption of tight junctions (3), eventually resulting in BCSFB leakage.