Inhibition of Microglial Phagocytosis Is Sufficient To Prevent Inflammatory Neuronal Death

Neuronal loss requires release of soluble inflammatory mediators and direct neuronal–microglial contact. A, LTA-induced neuronal loss over 3 d is prevented by separation of neurons/astrocytes from microglia by TWs, without increasing the densities of dying cells (necrotic cells not shown, because LME treatment leads to microglial lysis and chromatin release; cf. Fig. 1). ***p < 0.001 versus control, ^###p < 0.001 versus LTA. B, Microglial densities for experiment in A. *p < 0.05, **p < 0.01 versus respective controls. C, Separation of inflammatory soluble mediator release and direct microglial–neuronal contact using transwell cocultures (TW). First column from left, Microglia-depleted cultures were left untreated; second column, cultures were stimulated with LTA for 2 d, and LTA-activated microglia were then added for 6 h; third column, microglia-depleted cultures were exposed to nonactivated microglia on transwells for 2 d followed by addition of LTA-activated microglia for 6 h; fourth column, cultures were exposed to LTA-activated microglia on transwells for 2 d followed by LTA-activated microglia for 6 h. Delayed addition of LTA-activated microglia causes neuronal loss only if neurons were pre-exposed to soluble inflammatory mediators from LTA-activated transwell microglia. ^##p < 0.01 versus TW−. Data shown are means ± SEM for three or more independent experiments.

Inflammatory neuronal eat-me signaling is reversible and mediated by RONS. A, Removal of transwell microglia for 1 d after 3 d of LTA (50 μg/ml) stimulation leads to decreased neuronal loss on addition of activated microglia (cf. Fig. 2), indicating reversibility of neuronal eat-me signaling. ***p < 0.001 versus TW−, ^##p < 0.01 for TW LTA versus TW LTA − TW removed). B, Confocal micrographs of neurons below transwells (astrocytes lie in a layer underneath the focal plane) loaded with fluorescent PS (NBD-PS, green) at 3 d after LTA stimulation. Nuclei are counterstained with propidium iodide (red) after fixation. Note the reduced signal intensity of LTA-stimulated cultures (TW LTA) compared with control cultures (TW control) after quenching of exoplasmic NBD-PS, indicating increased PS exposure. Scale bars, 50 μm. C, LTA stimulation of transwell cultures for 3 or 4 d increases PS exposure (quantified as a decrease in neuronal NBD-PS fluorescence after quenching of exposed NBD-PS). Blocking endocytosis with cytochalasin D (Cyto D) has no effect on NBD-PS levels. The LTA-induced PS exposure is reversed by removing the transwell microglia or by scavenging superoxide (using SOD) or peroxynitrite (using FeTPPS) for 1 d after 3 d of LTA stimulation. For all bars, the NBD-PS fluorescence of quenched neurons is expressed as percentage of NBD-PS signal for untreated transwell cultures. D, LTA- (50 μg/ml) or LPS-induced (100 ng/ml) neuronal loss is prevented by delayed scavenging (added 2 d after LTA/LPS) of RONS using SOD, the SOD mimetic MnTBAP, or the peroxynitrite scavenger FeTPPS, or by inhibition of NO synthases using L-NAME, for 1 d. In contrast, catalase (CAT) has no effect. Neuronal density and death was quantified 3 d after LTA or LPS addition to mixed cultures. Microglial activation/proliferation was unaffected by all treatments (see Supplemental Table I). Data shown are means ± SEM for three or more independent experiments. **p < 0.01, ***p < 0.001 versus control; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 versus LTA or LPS.

Nontoxic levels of peroxynitrite cause reversible PS exposure and microglia-dependent neuronal loss. A, Low levels of peroxynitrite (10 μM) cause loss of neurons over 3 d without accumulation of dying cells in mixed cultures. B, In mixed cultures, peroxynitrite increases neuronal PS exposure (Annexin V⁺ cells) and chromatin-condensation between 1 h and 1 d after treatment, returning to baseline at 2 d, concomitant with a decrease in healthy cells (0 h indicates staining immediately after addition of peroxynitrite). C, Peroxynitrite (10 μM) does not cause significant neuronal death or loss in microglia-depleted cultures even 7 d after peroxynitrite addition (cf. A). D, In microglia-depleted cultures, peroxynitrite (10 μM) induces reversible chromatin condensation and PS exposure, but no loss of neurons. Data shown are means ± SEM for three or more independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001 versus control.

Inhibition of phagocytosis prevents LTA-, LPS-, peroxynitrite- and Aβ-induced neurodegeneration, leaving behind healthy neurons. A, Neuronal loss induced by LTA (50 μg/ml) or LPS (100 ng/ml) is reduced by inhibiting the PS–MFG-E8–vitronectin receptor system. Significant neuroprotection is provided by: 1) preventing PS recognition through addition of C2A domain, 2) blocking MFG-E8 bridging of PS-exposing cells and the vitronectin receptor on microglia using a MFG-E8 blocking Ab, or 3) using the vitronectin receptor-specific blocking peptide cRGD to prevent MFG-E8 recognition. In contrast, a control IgG and cRAD have no effect. B, Protection provided by cRGD (50 μM) against LPS (100 ng/ml)-induced neuronal loss does not lead to accumulation of caspase-3⁺ cells 3 d after stimulation. C, Loading with TMRM (3 nM) indicates that the plasmalemmal and the mitochondrial membrane potential are intact in neurons protected from LPS (100 ng/ml)-induced neuronal loss by cRGD (50 μM). Dark bars represent the number of healthy neurons as a percentage of that in the untreated control culture; light bars represent the total number of neurons positive for TMRM as percentage of that in the untreated control culture. Thus, in each treatment, virtually all neurons present are TMRM⁺. D, Treatment of mixed cultures with nontoxic levels of peroxynitrite and SIN-1 leads to significant neuronal loss after 3 d. This does not occur after decomposition of either reagent in culture medium (“decomposed”). The SIN-1 effect is mediated by peroxynitrite as shown by prevention of neuronal loss with the peroxynitrite scavenger FeTPPS. The vitronectin receptor antagonist, cRGD, protects against neuronal loss induced by low peroxynitrite (5 μM), and partially protects against intermediate peroxynitrite (10 μM) and SIN-1 (50 μM), but not high peroxynitrite (20 μM), which induces significant cell death by necrosis and apoptosis. E, Treatment of mixed cultures with the β-amyloid peptide (25–35) (Aβ[25–35], 250 nM) leads to significant neuronal loss over 3 d, whereas the reverse peptide Aβ(35–25) has no effect on neuronal density. Aβ(25–35)-induced neuronal loss is not prevented by incubation with polymyxin B (PMXN B), excluding LPS contamination. In contrast, elimination of microglia using LME treatment, delayed addition of the peroxynitrite scavenger FeTPPS (2 d after stimulation), or cotreatment with the vitronectin receptor antagonist, cRGD, significantly prevents Aβ(25–35) induced neuronal loss. The control peptide cRAD, in contrast, has no effect. Gray lines indicate 100% and the approximate level of neuronal loss for ease of comparison. Data shown are means ± SEM for three or more independent experiments. **p < 0.01, ***p < 0.001 compared with control; ^#p < 0.05, ^##p < 0.01 compared with LTA/LPS/peroxynitrite/SIN-1/Aβ(25–35).