Activated microglia mediate synapse loss and short-term memory deficits in a mouse model of transthyretin-related oculoleptomeningeal amyloidosis

Abstract

Oculoleptomeningeal amyloidosis (OA) is a fatal and untreatable hereditary disease characterized by the accumulation of transthyretin (TTR) amyloid within the central nervous system. The mechanisms underlying the pathogenesis of OA, and in particular how amyloid triggers neuronal damage, are still unknown. Here, we show that amyloid fibrils formed by a mutant form of TTR, A25T, activate microglia, leading to the secretion of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and nitric oxide. Further, we found that A25T amyloid fibrils induce the activation of Akt, culminating in the translocation of NFκB to the nucleus of microglia. While A25T fibrils were not directly toxic to neurons, the exposure of neuronal cultures to media conditioned by fibril-activated microglia caused synapse loss that culminated in extensive neuronal death via apoptosis. Finally, intracerebroventricular (i.c.v.) injection of A25T fibrils caused microgliosis, increased brain TNF-α and IL-6 levels and cognitive deficits in mice, which could be prevented by minocycline treatment. These results indicate that A25T fibrils act as pro-inflammatory agents in OA, activating microglia and causing neuronal damage.

Figure 1

A25T fibrils activate microglia in vitro and in vivo. A25T fibrils (Fib A25T) were fluorescently labeled with acrylodan (pseudocolored in green in panels a–d) and incubated with microglia for 24 h, which nuclei (DNA) were labeled in red with ethidium homodimer-1. (a–d) Two-photon microscopy of microglia shows internalization of fluorescent A25T fibrils (green) together with the cells (DIC; a) and inside the cells (b and inset in panel B). A25T fibrils internalization was inhibited using 2.5 μM cytochalasin D (+Cyt-D) (d) when compared with untreated cells (control; c). Inset in (d) shows microglia incubated with soluble A25T, which is not internalized by microglia in 24 h. Scale bars are 25 μm for panels A-B, 100 μm for panels c and b and 50 μm for inset in panel d. We also measured TNF-α (e), IL-6 (f) and (g) NO (measured as nitrite using Griess reagents) in culture supernatants. As controls, PBS and soluble A25T (Sol A25T) at 1 μM were also incubated with microglia. As positive control, LPS at 100 ng/ml was incubated with microglia. Statistical analysis was performed in three independent experiments using one-way ANOVA with Tukey's test, and ***P< 0.001, **P<0.01 and *P<0.05. In panel e, ** represents the comparison between PBS and Fib A25T and * represents the comparison between Sol A25T and Fib A25T. In panel f, * represents the comparison between PBS and Fib A25T and ** represents the comparison between Sol A25T and Fib A25T. (h–r) Two-month-old Swiss mice were i.c.v. injected with A25T fibrils or vehicle (PBS). After 24 h, (h–k) A25T fibrils- and (l–o) vehicle-injected mice were killed and brain sections were stained with DAPI (blue; h and l), anti-F4/80 (green, i and m) and anti-human TTR (Fib A25T; red, j and n), and then analyzed using confocal microscopy. Merged images are shown in k and o. Scale bars are 24 μm for all images. After 4 h, mice were euthanized and brain homogenates were analyzed for TNF-α (p) and IL-6 (q). Statistical analysis of panel p and q was performed using Student's t-test were *P<0.05, **P<0.01. (r) Iba-1 levels were analyzed in lysates from A25T fibrils- and vehicle-injected mice by western blotting. Statistical analysis was performed using Student's t-test were *P<0.05

Figure 2

A25T fibrils induce microglia activation via Akt phosphorilation at Ser473, GSK-3β phosphorilation at Ser9 and NFκB translocation to cell nucleus. Primary microglia cells were incubated with 1 μM of fibrillar A25T (Fib A25T) or PBS for 30 min. Cells were then lysed and protein lysates analyzed by (a) western blotting using anti-phosphorilated Akt (pAkt) at Ser473 and anti-phosphorilated GSK-3β (pGSK-3β) at Ser9. Levels of phosphorilated protein (b; % of pAkt/Akt) or (c; % of pGSK-3β/GSK-3β) were analyzed relative to PBS (control; 100%). Statistical analysis was performed in three independent experiments using Student's t-test; *P<0.05. Panels d–p depict NFκB translocation to the nucleus: d–f shows microglia incubated with PBS, soluble A25T (Sol A25T; g–i), fibrillar A25T (Fib A25T; j–l) and LPS (m–o). NFκB+ nuclei/total of nuclei cells were quantified in p. Statistical analysis was performed in three independent experiments using one-way ANOVA, with *P<0.05. Scale bars are 25 μm for panels d–o

Figure 3

CM from A25T fibrils-activated microglia induce synapse loss in primary cortical neurons. A25T fibrils or soluble A25T at 1 μM were incubated for 48 h with microglia cells and then CM (Fib A25T CM and Sol A25T CM) was transferred to primary neurons for 3 h. As controls, we used CM from PBS- or LPS (100 ng/ml)-activated microglia. Cells were fixed and immunostained with antibodies anti-PSD-95 (red) and anti-synaptophysin (green), and the colocalization (puncta; yellow) of both proteins suggest the presence of a functional synapse. Images show a representative neuron incubated with (a) PBS CM, (b) Sol A25T CM, (c) Fib A25T CM, (d) LPS CM. (e) Quantification of colocalization of PSD-95 and synaptophysin (number of puncta per image). Each experiment was performed in duplicates and 12–20 images were captured, quantified for colocalization and the mean of all images was plotted for each experiment. Statistical analysis was performed in three independent experiments using Kruskal–Wallis test and Dunn's post hoc test, and ***P<0.01 when PBS CM and Agg A25T CM were compared and **P<0.01 when Sol A25T CM and Agg A25T CM were compared. Scale bars are 25 μm for all panels

Figure 4

CM from A25T fibril-activated microglia induce neuron death. Fibrillar A25T (Fib A25T) or soluble A25T (Sol A25T) were incubated with primary microglia cells for 48 h to generate fibrillar A25T-conditioned media (Fib A25T CM) or soluble A25T-CM (Sol A25T CM) and then, CM were incubated with primary cortical neuron cultures for 48 h. Cell viability was assessed using Live/Dead assay, where live cells are stained in green and dead cells in red (a–e). Also as controls, PBS and 100 ng/ml LPS were incubated first with microglia to generate CMs (PBS CM; A and LPS CM; d) and then incubated with neurons. To test microglia-mediated cell death, (b) Sol A25T CM or (c) Fib A25T CM was incubated with neurons for 48 h. Panel (e) show the percentage (%) of live cells compared with medium alone (considered as 100% viability; not shown) and statistical analysis was performed in three independent experiments using one-way ANOVA, with Tukey's test, and **P<0.01, ***P<0.001. Scale bars are 50 μm

Figure 5

CM from A25T fibril-activated microglia induce caspase-3 activation and fragmentation of DNA in neurons. A25T fibrils (Fib A25T CM) or soluble A25T (Sol A25T CM) at 1 μM were incubated with primary microglia for 48 h and CM was transferred to cortical neurons for 24 h. As controls, we used conditioned media from PBS- or LPS (100 ng/ml)-activated microglia. Caspase-3 activation was analyzed using an antibody anti-cleaved caspase-3 (red, a–e) and DNA fragmentation was monitored by TUNEL assay (f–j). Cells were also stained with Hoescht (blue; a–d and f–i). In (e) cleaved caspase-3-positive cells were quantified and compared with total cells (Hoescht-positive cells) and % of cleaved caspase-3-positive cells were plotted relative to neurons incubated with LPS CM. In (j) TUNEL-positive cells were quantified and compared to total cells (Hoescht-positive cells) and % of TUNEL-positive cells was plotted relative to positive control, DNAse I treated cells (not shown; considered 100%). Statistical analysis was performed in three independent experiments using one-way ANOVA, with Tukey's test, and ***P<0.001 and **P<0.01. In panel e, ** represents the comparison between PBS and Fib A25T and * represents the comparison between Sol A25T and Fib A25T. In panel F, ** represents the comparison between PBS and Fib A25T and *** represents the comparison between Sol A25T and Fib A25T. Scale bars are 100 μm for panels a–d and 25 μm for panels (e–h)

Figure 6

Minocycline prevented A25T fibrils-induced short-term memory loss in the novel object recognition test. Two-month-old Swiss mice were pretreated with 50 mg/kg/day minocycline for 3 consecutive days and then i.c.v. injected with A25T fibrils or vehicle (PBS). After 24 h, animals were subjected to the novel object recognition test in which a familiar object (A) was substituted by a novel object (C). Exploratory activity was measured and % of exploration was analyzed in all animals. Statistical analysis was performed using one-sample t-test applying a hypothetical value of 50.0; ***P<0.001 and **P<0.05