Synergistic effects of the Aβ/fibrinogen complex on synaptotoxicity, neuroinflammation, and blood-brain barrier damage in Alzheimer's disease models

Abstract

Introduction: Alzheimer's disease (AD) is characterized by amyloid-beta (Aβ), hyperphosphorylated tau, chronic neuroinflammation, blood-brain barrier (BBB) damage, and synaptic dysfunction, leading to neuronal loss and cognitive deficits. Vascular proteins, including fibrinogen, extravasate into the brain, further contributing to damage and inflammation. Fibrinogen's interaction with Aβ is well-established, but how this interaction contributes to synaptic dysfunction in AD is unknown.

Methods: Organotypic hippocampal cultures (OHC) were exposed to Aβ42 oligomers, fibrinogen, or Aβ42/fibrinogen complexes. Synaptotoxicity was analyzed by Western blot. Aβ42 oligomers, fibrinogen, or their complexes were intracerebroventricularly injected into mice. Histopathological AD markers, synaptotoxicity, neuroinflammation, and vascular markers were observed by Western blot and immunofluorescence.

Results: Aβ42/fibrinogen complexes led to synaptic loss, tau181 phosphorylation, neuroinflammation, and BBB disruption, independent of Mac1/CD11b receptor signaling. Blocking Aβ42/fibrinogen complex formation prevented synaptotoxicity.

Discussion: These findings indicate that the Aβ42/fibrinogen complex has a synergistic impact on hippocampal synaptotoxicity and neuroinflammation.

Highlights: Fibrinogen binds to the central region of Aβ, forming a plasmin-resistant complex. The Aβ/fibrinogen complex induces synaptotoxicity, inflammation, and BBB disruption. Synaptotoxicity induced by the complex is independent of Mac1 receptor signaling.

Keywords: Alzheimer's disease; amyloid‐beta; blood‐brain barrier; fibrinogen; neuroinflammation; synaptotoxicity.

FIGURE 1

The Aβ₄₂/fibrinogen complex induces synergistic synaptotoxicity in mouse organotypic hippocampal slice cultures (OHCs). (A)–(C) OHCs were incubated with vehicle (control; 0.005% DMSO in PBS), low‐dose Aβ42 oligomers (150 nM), high‐dose Aβ42 oligomers (500 nM), fibrinogen (FBG, 50 nM), or Aβ42/FBG complex (150 and 50 nM, respectively) for 24 h and then collected for biochemical analysis. (A) Representative Western blot of SYP and post‐synaptic density 95 (PSD‐95) levels in OHC lysates. Protein analyses were normalized to β‐actin. Quantification shows a reduction in (B) SYP and (C) PSD‐95 in OHCs treated with the Aβ42/FBG complex (red) or the high dose of Aβ alone (blue). (D)–(F) OHCs were treated with vehicle (control), low‐dose Aβ42 oligomers (150 nM), FBG (50 nM), or Aβ42/FBG complex with or without 4G8 anti‐Aβ antibody for 24 h and then collected and prepared for biochemical analysis. (D) Representative Western blot of SYP and PSD‐95 levels in OHC lysates. Quantification shows a reduction of (E) SYP and (F) PSD‐95 in OHCs treated with Aβ42/FBG complex only (red). Slices treated with Aβ42/fibrinogen and 4G8 (blue) showed no changes in synapse protein levels. n = 3 independent experiments. More precisely, each well/insert contained six hippocampal slices total from three different animals (two slices per animal), and there were three wells per treatment group. The result from each well generated a single data point, although it represented three animals. Each experiment encompassed nine mice but only three data points. Data were analyzed by one‐way ANOVA with Tukey's post hoc analysis. Aβ, amyloid‐beta; ANOVA, analysis of variance; DMSO, dimethyl sulfoxide; PBS, phosphate buffered saline; SYP, synaptophysin. ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001

FIGURE 2

The Aβ42/fibrinogen complex reduces SYP and PSD‐95 levels and induces tau181 phosphorylation (p‐tau181) in the hippocampus of WT mice. WT mice were injected ICV with aCSF, Aβ42 (1.5 or 3.0 µM), fibrinogen (FBG, 7.5 µM), or the preformed Aβ42/FBG complex using low‐dose (1.5 µM) Aβ42. Three days post‐injection, brains were processed for Western blotting. (A) Representative Western blot of SYP and PSD‐95 levels in hippocampal homogenates. Protein analyses were normalized to β‐actin. β‐actin bands shown are the same as in Figure 5F; this membrane and sample set was probed for multiple proteins. Quantification shows a reduction of (B) SYP and (C) PSD‐95 in hippocampal homogenates of mice injected with the Aβ42/FBG complex compared to other treatments (red). (D) Representative Western blot of total tau and phosphorylated tau181 (p‐tau181) levels in the same hippocampal homogenates. (E) Quantification of Western blots indicates that ICV injection of the Aβ42/FBG complex led to increased tau181 phosphorylation in the WT mouse hippocampus compared to other treatments (red). Statistical analyses were performed using one‐way ANOVA followed by Tukey's post‐hoc test. Bar graphs represent mean ± SEM. n = 3–4 mice/group. Aβ, amyloid‐beta; aCSF, artificial cerebrospinal fluid; ANOVA, analysis of variance; ICV, intracerebroventricularly; PSD, postsynaptic density protein; SEM, structural equation modeling; SYP, synaptophysin; WT, wild‐type. ^* p < 0.5, ^** p < 0.01, ^**** p < 0.0001

FIGURE 3

The Aβ42/FBG complex increases inflammatory activity in the hippocampus of WT mice. WT mice were injected ICV with aCSF, the pre‐formed Aβ42/FBG complex (1.5 or 7.5 µM, respectively), Aβ42 oligomers (1.5 or 3.0 µM), or FBG (7.5 µM). After 3 days, brains were processed for immunostaining. Hippocampal sections were stained for (A) CD68 (red) or (B) GFAP. (C), (D) There was significantly more CD68 and GFAP staining in the CA1 region of the hippocampus of the Aβ42/FBG complex‐injected mouse group compared to all other treatment groups. Scale bar, 50 µm. Data were analyzed by one‐way ANOVA with Tukey's post hoc analysis. n = 4/group. Aβ, amyloid‐beta; aCSF, artificial cerebrospinal fluid; ANOVA, analysis of variance; FBG, fibrinogen; GFAP, glial fibrillary acidic protein; ICV, intracerebroventricularly; WT, wild‐type. ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001.

FIGURE 4

The Aβ42/fibrinogen complex induces synaptotoxicity independent of Mac‐1 receptor signaling. (A)–(C) OHC slices from Mac‐1 KO mice were incubated with vehicle (0.005% DMSO in PBS; control), high or low doses of Aβ42 oligomers (500 or 150 nM), FBG (50 nM), or the pre‐formed Aβ42/FBG complex (150 and 50 nM, respectively) for 24 h. Following treatment, OHCs were washed, lysed, and prepared for biochemical analysis. (A) Representative Western blot of SYP and PSD‐95 levels in Mac‐1 KO OHC lysate. Quantification shows a reduction of (B) SYP and (C) PSD‐95 in Mac‐1 KO slices treated with Aβ42/FBG complex only (red). n = 5 independent experiments/5 wells per treatment; each experiment utilized 3 mice; 15 mice in total. (D)–(F) Mac‐1 KO mice were injected ICV with aCSF, Aβ42/FBG complex (1.5 and 7.5 µM, respectively), Aβ42 oligomer (1.5 or 3.0 µM), or FBG (7.5 µM). Three days post‐injection, brains were processed for Western blotting. (D) Representative Western blot of SYP and PSD‐95 levels in hippocampal homogenates. Protein analyses were normalized to β‐actin. Quantification shows a reduction of (E) SYP and (F) PSD‐95 in hippocampal homogenates from mice injected with Aβ42/FBG complex (red). n = 5 mice/group. Statistical analyses were performed using one‐way ANOVA followed by Tukey's post‐hoc test. Aβ, amyloid‐beta; aCSF, artificial cerebrospinal fluid; ANOVA, analysis of variance; DMSO, dimethyl sulfoxide; FBG, fibrinogen; ICV, intracerebroventricularly; KO, knockout; OHC, organotypic hippocampal slice culture; PBS, phosphate buffered saline; PSD, postsynaptic density protein; SYP, synaptophysin; WT, wild‐type. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001,^**** p < 0.0001.

FIGURE 5

The Aβ42/FBG complex induces BBB disruption and extravasation in the hippocampus. WT mice were ICV injected with aCSF, Aβ42 /FBG complex (1.5 and 7.5 µM, respectively), Aβ42 oligomers (1.5 or 3.0 µM), or FBG (7.5 µM). Three days post‐injection, brains were processed for Western blotting. Hippocampal homogenates were analyzed by Western blot for CD‐31, occludin, ZO‐1, and AQP‐4. (A) Representative Western blot. (B)–(E) Quantification of CD31, occludin, ZO‐1, and AQP‐4 levels in hippocampal homogenates, normalized to β‐actin. The Aβ42/FBG complex led to a synergistic decrease in all proteins examined (red). (F) Representative Western blot of hippocampal homogenates probed for mouse fibrinogen and albumin. β‐actin bands shown are the same as in Figure 2A; this membrane and sample set was probed for multiple proteins. (G), (H) Quantification of fibrinogen and albumin normalized to β‐actin. The Aβ42/FBG complex induced significantly more extravasation of blood proteins into the mouse hippocampus compared to all other treatment groups (red). Statistical analyses were performed using one‐way ANOVA followed by Tukey's post‐hoc test. Bar graphs represent mean ± SEM. Aβ, amyloid‐beta; aCSF, artificial cerebrospinal fluid; ANOVA, analysis of variance; BBB, blood–brain barrier; FBG, fibrinogen; ICV, intracerebroventricularly; PSD, postsynaptic density protein; SEM, structural equation modeling; SYP, synaptophysin; WT, wild‐type. n = 3–4/group. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001.