Glia as a therapeutic target: selective suppression of human amyloid-beta-induced upregulation of brain proinflammatory cytokine production attenuates neurodegeneration

Abstract

A corollary of the neuroinflammation hypothesis is that selective suppression of neurotoxic products produced by excessive glial activation will result in neuroprotection. We report here that daily oral administration to mice of the brain-penetrant compound 4,6-diphenyl-3-(4-(pyrimidin-2-yl)piperazin-1-yl)pyridazine (MW01-5-188WH), a selective inhibitor of proinflammatory cytokine production by activated glia, suppressed the human amyloid-beta (Abeta) 1-42-induced upregulation of interleukin-1beta, tumor necrosis factor-alpha, and S100B in the hippocampus. Suppression of neuroinflammation was accompanied by restoration of hippocampal synaptic dysfunction markers synaptophysin and postsynaptic density-95 back toward control levels. Consistent with the neuropathophysiological improvements, MW01-5-188WH therapy attenuated deficits in Y maze behavior, a hippocampal-linked task. Oral MW01-5-188WH therapy begun 3 weeks after initiation of intracerebroventricular infusion of human Abeta decreased the numbers of activated astrocytes and microglia and the cytokine levels in the hippocampus without modifying amyloid plaque burden or altering peripheral tissue cytokine upregulation in response to an in vivo inflammatory challenge. The results provide a novel integrative chemical biology proof in support of the neuroinflammation hypothesis of disease progression, demonstrate that neurodegeneration can be attenuated independently of plaque modulation by targeting innate brain proinflammatory cytokine responses, and indicate the feasibility of developing efficacious, safe, and selective therapies for neurodegenerative disorders by targeting key glial activation pathways.

Figure 1.

Ex vivo inhibition of proinflammatory cytokine production by MW01-5-188WH and stability after incubation with rat liver microsomes. A, Structure of MW01-5-188WH. Concentration-dependent inhibition by MW01-5-188WH of LPS-induced increases of IL-1β (B) and TNF-α (C) levels in the BV-2 microglial cell line is shown. D, LPS-stimulated accumulation of the NO metabolite nitrite was not inhibited by MW01-5-188WH at concentrations up to 33 μm. MW01-5-188WH (188) does not suppress LPS-induced production of iNOS (E) or COX-2 (F) in activated BV-2 cells. The compound also does not block Aβ-induced apoE upregulation (G) in activated rat primary glia cultures. Cultures were treated with control buffer (C) or activated in the absence (A) or presence (A+188) of 7.5 μm MW01-5-188WH, then analyzed by Western blots. H, Amount of remaining MW01-5-188WH (1 μm) after 30 and 120 min incubation with rat liver microsomes. MW01-5-188WH is stable at 30 min but is able to be metabolized by 120 min. Error bars represent SEM.

Figure 2.

MW01-5-188WH is readily detected in the plasma and the brain after a single oral dose administration and does not suppress peripheral tissue inflammatory responses or cause liver injury after chronic oral administration. C57BL/6 mice were administered MW01-5-188WH (2.5 mg/kg) by oral gavage, blood and brain processed at different times after administration, and compound levels in plasma and brain determined as described in Materials and Methods. MW01-5-188WH rapidly appears in plasma (A) and brain (B), reaches a peak at 15 min, and then slowly declines to basal levels by 120 min. Data are the mean± SEM from three to six mice at each time point. MW01-5-188WH does not inhibit increased production of IL-1β (C) and TNF-α (D) in the serum but does suppress the cytokine response in the brains from the same mice (E, F). Mice (n = 3-6 per group) were administered by oral gavage either diluent or MW01-5-188WH (2.5 mg/kg) once daily for 2 weeks and then challenged with LPS (10 mg/kg, i.p.) for 6 h. Control mice were injected with saline. IL-1β and TNF-α levels in the serum and in brain supernatants were determined. Data represent mean ± SEM. ^***p < 0.001, significantly different from diluent. Daily oral administration of diluent (G) or MW01-5-188WH (H) (2.5 mg/kg) does not result in any histological liver toxicity. Representative liver sections from mice treated as in C-F were stained with hematoxylin and eosin. Scale bar, 125 μm. 188, MW01-5-188WH.

Figure 3.

Oral administration of MW01-5-188WH suppresses human Aβ-induced upregulation of proinflammatory cytokine production in mouse hippocampus without detectable effect on the profile of nitrotyrosine-labeled neurons or on amyloid plaque deposition. A schematic of the experimental paradigm is shown. Mice were subjected to vehicle infusion (Control) or human oligomeric Aβ_1-42 infusion (No treatment) for 4 weeks. At 3 weeks after the start of infusion and continuing once daily for 2 weeks thereafter, mice were administered by oral gavage either 2.5 mg/kg MW01-5-188WH (188-treated) or diluent (No treatment). MW01-5-188WH treatment resulted in significant suppression of the Aβ-induced increase in IL-1β (A), TNF-α (B), and S100B (C) levels in hippocampal supernatants (n = 10 mice per group). MW01-5-188WH treatment also decreased the number of GFAP-positive activated astrocytes (D) and F4/80-positive microglia (E) in hippocampus. F, MW01-5-188WH treatment did not alter the profile of nitrotyrosine-stained neurons, an indicator of oxidative stress-linked injury. Representative micrographs are shown for hippocampus sections stained for nitrotyrosine-positive neurons from vehicle-infused mice, Aβ-infused mice orally administered diluent (No treatment), and Aβ-infused mice orally administered MW01-5-188WH (188-treated) for 2 weeks. Scale bar, 25 μm. The number of amyloid plaques (G) or the area occupied by plaques (H) is not altered by MW01-5-188WH therapy. Data are mean ± SEM of n = 5 mice per group. Significantly different from Aβ infused: ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 4.

Oral administration of MW01-5-188WH attenuates hippocampal synaptic dysfunction and hippocampus-linked behavioral deficits. A schematic of the experimental paradigm is shown. Mice were treated as in Figure 3. MW01-5-188WH administration significantly attenuated Aβ-induced loss of synaptophysin and PSD-95 and the behavioral deficit in the Y maze. Levels of the presynaptic protein synaptophysin (A) and the postsynaptic protein PSD-95 (B) were measured in hippocampal supernatants from vehicle-infused mice (Control), Aβ-infused mice administered diluent (No treatment), and Aβ-infused mice administered MW01-5-188WH (188-treated) at 2.5 mg/kg by oral gavage once daily for 2 weeks. C, Spontaneous alternation of mice in the Y maze, a hippocampus-dependent spatial learning task, was measured for 10 d during the seventh and eighth week after the start of Aβ infusion. Data are mean ± SEM of n = 5-10 mice per group. Significantly different from Aβ infused: ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.