Extracellular tau stimulates phagocytosis of living neurons by activated microglia via Toll-like 4 receptor-NLRP3 inflammasome-caspase-1 signalling axis

Abstract

In tauopathies, abnormal deposition of intracellular tau protein followed by gradual elevation of tau in cerebrospinal fluids and neuronal loss has been documented, however, the mechanism how actually neurons die under tau pathology is largely unknown. We have previously shown that extracellular tau protein (2N4R isoform) can stimulate microglia to phagocytose live neurons, i.e. cause neuronal death by primary phagocytosis, also known as phagoptosis. Here we show that tau protein induced caspase-1 activation in microglial cells via 'Toll-like' 4 (TLR4) receptors and neutral sphingomyelinase. Tau-induced neuronal loss was blocked by caspase-1 inhibitors (Ac-YVAD-CHO and VX-765) as well as by TLR4 antibodies. Inhibition of caspase-1 by Ac-YVAD-CHO prevented tau-induced exposure of phosphatidylserine on the outer leaflet of neuronal membranes and reduced microglial phagocytic activity. We also show that suppression of NLRP3 inflammasome, which is down-stream of TLR4 receptors and mediates caspase-1 activation, by a specific inhibitor (MCC550) also prevented tau-induced neuronal loss. Moreover, NADPH oxidase is also involved in tau-induced neurotoxicity since neuronal loss was abolished by its pharmacological inhibitor. Overall, our data indicate that extracellular tau protein stimulates microglia to phagocytose live neurons via Toll-like 4 receptor-NLRP3 inflammasome-caspase-1 axis and NADPH oxidase, each of which may serve as a potential molecular target for pharmacological treatment of tauopathies.

Figure 1

Tau induces caspase-1 activation in microglial cells via TLR4 receptors and neutral sphingomyelinase (nSMase). (A) representative confocal microscopy images of caspase-1 labelling in untreated (control) or treated with 3 µM tau for 24 h neuronal glial-co-cultures. Neuronal cell can be identified in differential interference contrast (DIC) images. Cell nuclei were labelled with Hoechst 33342 (blue), microglial cells with isolectin B₄-AlexaFluor568 (red) and activated caspase-1 with FamFlica reagent (green). Scale bars, 10 µm. (B) FamFlica fluorescence intensity in microglial cells. Pure cell cultures were incubated with 3 µM tau for 24 h with or without 1 µg/mL anti-TLR4 antibody or 11 µM nSMase inhibitor GW4869. FamFlica fluorescence intensity in tau-treated (with/without inhibitors) groups is expressed as the percentage of fluorescence intensity in the control group (untreated), which were considered as 100%. Data are presented as means ± SE for 3 independent experiments; *p < 0.05 versus untreated control, ^#p < 0.05, ^##p < 0.01 versus tau treated cultures.

Figure 2

Tau-induced neurotoxicity is prevented by caspase-1 inhibitors. (A) representative images of untreated (control) and tau treated (3 µM for 48 h) neuronal glial-co-cultures with or without 1 µM YVAD-CHO. In phase contrast images, neuronal cell can be identified by characteristic shape and morphology. Cell nuclei were labelled with Hoechst 33342: cells with homogeneously stained nuclei (blue) were considered as viable, cells with condensed or fragmented nuclei as apoptotic. Necrotic cells were stained with propidium iodide (red) and microglial cells with isolectin B₄-AlexaFluor488 (green). Scale bars, 100 µm. (B) neuronal viability, (C) neuronal number and (D) microglial number in neuronal-glial co-cultures after incubation with 3 µM tau for 48 h with and without 1 µM YVAD-CHO or 200 nM VX-765. Neuronal viability is expressed as ratio of live and dead (necrotic and apoptotic) cells in a population. Number of neurons and microglia in tau-treated (with/without caspase-1 inhibitors) groups is expressed as the percentage of the total number of appropriate cells in the control (untreated) group, which were considered as 100%. (E) interleukin (IL)-1β and -18 level in mixed cell culture medium after incubation with 3 µM tau for 24 h. Data are presented as means ± SE for 5–6 independent experiments; ***p < 0.001 versus untreated control, ^#p < 0.05, ^###p < 0.001 versus tau treated cultures.

Figure 3

Inhibition of caspase-1 suppresses tau stimulated phosphatidylserine exposure and microglial phagocytic activity. (A) phagocytosis of latex beads in pure microglial cell cultures. Changes of latex bead uptake in tau-treated groups (with/without YVAD-CHO) were expressed as percentage of fluorescence intensity in the control (untreated) group, which were considered as 100%. (B) representative images demonstrating the uptake of latex beads (red) by microglial cells. (C) phosphatidylserine exposure evaluated by Annexin V-Cy3.18 fluorescence intensity in neuronal glial co-cultures. Changes in fluorescence intensity of AnnexinV-Cy3.18 in tau-treated groups (with/wthout YVAD-CHO) were expressed as percentage of fluorescence intensity in the control (untreated) group, which were considered as 100%. (D) representative images of phosphatidylserine exposure. Neuronal external phosphatidylserine was labelled with Annexin V-Cy3.18 conjugate (red), cell nuclei was stained with Hoescht33342 (blue). Cell cultures were treated with 3 µM tau for 24 h with or without 1 µM YVAD-CHO. Scale bars, 10 µm. Data are presented as means ± SE for 3 independent experiments; ***p < 0.001 versus untreated control, ^#p < 0.05, ^##p < 0.01 versus tau-treated cultures.

Figure 4

Antibodies blocking TLR4 protect against tau induced neuronal loss. (A) neuronal viability, (B) neuronal number, (C) microglial number. Neuronal viability is expressed as ratio of live and dead (necrotic and apoptotic) cells in a population. Number of neurons and microglia in tau-treated (with/without caspase-1 inhibitors) groups is expressed as the percentage of the total number of appropriate cells in the control (untreated) group, which were considered as 100%. Neuronal-glial co-cultures were incubated with 3 µM tau for 48 h with and without 1 µg/mL anti-TLR4 antibody or 1 µg/ml IgG. In (A–C) data are presented as means ± SE for 5–6 independent experiments; **p < 0.01, ***p < 0.001 versus untreated control, ^###p < 0.001 versus tau treated cultures. (D) representative images of untreated (control) and tau treated (3 µM for 48 h) neuronal glial-co-cultures with or without 1 µg/mL anti-TLR4 antibody. In phase contrast images, neuronal cell can be identified by characteristic shape and morphology. Cell nuclei were labelled with Hoechst 33342: cells with homogeneously stained nuclei (blue) were considered as viable, cells with condensed or fragmented nuclei as apoptotic. Necrotic cells were stained with propidium iodide (red) and microglial cells with isolectin B₄-AlexaFluor488 (green). Scale bars, 100 µm.

Figure 5

NLPR3 inflammasome inhibitor protects against tau-induced neuronal loss. (A) neuronal viability, (B) neuronal number, (C) microglial number. Neuronal viability is expressed as ratio of live and dead (necrotic and apoptotic) cells in a population. Number of neurons and microglia in tau-treated (with/without NLPR3 inflammasome inhibitor MCC950) groups expressed as the percent of the total number of appropriate cells in the control (untreated) group, which were considered as 100%. Neuronal-glial co-cultures were incubated with 3 µM tau for 48 h with and without 1 µM MCC950. In (A–C) data are presented as means ± SE for 3 independent experiments; ***p < 0.001, ** p < 0.01 versus untreated control, ^###p < 0.001, ^# p < 0.05 versus tau-treated cultures. (D) representative images of untreated (control) and tau treated (3 µM for 48 h) neuronal glial-co-cultures with or without 1 µM MCC950. In phase contrast images, neuronal cell can be identified by characteristic shape and morphology. Cell nuclei were labelled with Hoechst 33342: cells with homogeneously stained nuclei (blue) were considered as viable, cells with condensed or fragmented nuclei as apoptotic. Necrotic cells were stained with propidium iodide (red) and microglial cells with isolectin B₄-AlexaFluor488 (green). Scale bars, 100 µm.

Figure 6

NADPH oxidase inhibitor supresses tau-induced neurotoxicity. (A) neuronal viability, (B) neuronal number, (C) microglial number. Neuronal viability is expressed as ratio of live and dead (necrotic and apoptotic) cells in a population. Number of neurons and microglia in tau-treated (with/without NADPH oxidase inhibitor GSK2795039) groups expressed as the percentage of the total number of appropriate cells in the control (untreated) group, which were considered as 100%. Neuronal-glial co-cultures were incubated with 3 µM tau for 48 h with and without 1 µM GSK2795039. In A, B and C data are presented as means ± SE for 3–5 independent experiments; **p < 0.01, ***p < 0.001 versus untreated control, ^#p < 0.05, ^###p < 0.001 versus tau-treated cultures. (D) representative images of untreated (control) and tau treated (3 µM for 48 h) neuronal glial-co-cultures with or without 1 µM GSK2795039. In phase contrast images, neuronal cell can be identified by characteristic shape and morphology. Cell nuclei were labelled with Hoechst 33342: cells with homogeneously stained nuclei (blue) were considered as viable, cells with condensed or fragmented nuclei as apoptotic. Necrotic cells were stained with propidium iodide (red) and microglial cells with isolectin B₄-AlexaFluor488 (green). Scale bars, 100 µm.

Figure 7

Possible mechanism of tau-induced phagoptosis. Tau protein activates microglial TLR4 receptors and neutral sphingomyelinase (nSMase) which in turn induce caspase-1 and NADPH oxidase 2 (NOX2) activation, which leads to reactive oxygen production (ROS). ROS may lead to phosphatidylserine exposure on neurons. This can be recognized by microglia triggering microglial phagocytosis of stressed-but-viable neurons.