Early stage drug treatment that normalizes proinflammatory cytokine production attenuates synaptic dysfunction in a mouse model that exhibits age-dependent progression of Alzheimer's disease-related pathology

Abstract

Overproduction of proinflammatory cytokines in the CNS has been implicated as a key contributor to pathophysiology progression in Alzheimer's disease (AD), and extensive studies with animal models have shown that selective suppression of excessive glial proinflammatory cytokines can improve neurologic outcomes. The prior art, therefore, raises the logical postulation that intervention with drugs targeting dysregulated glial proinflammatory cytokine production might be effective disease-modifying therapeutics if used in the appropriate biological time window. To test the hypothesis that early stage intervention with such drugs might be therapeutically beneficial, we examined the impact of intervention with MW01-2-151SRM (MW-151), an experimental therapeutic that selectively attenuates proinflammatory cytokine production at low doses. MW-151 was tested in an APP/PS1 knock-in mouse model that exhibits increases in AD-relevant pathology progression with age, including increases in proinflammatory cytokine levels. Drug was administered during two distinct but overlapping therapeutic time windows of early stage pathology development. MW-151 treatment attenuated the increase in microglial and astrocyte activation and proinflammatory cytokine production in the cortex and yielded improvement in neurologic outcomes, such as protection against synaptic protein loss and synaptic plasticity impairment. The results also demonstrate that the therapeutic time window is an important consideration in efficacy studies of drugs that modulate glia biological responses involved in pathology progression and suggest that such paradigms should be considered in the development of new therapeutic regimens that seek to delay the onset or slow the progression of AD.

Figure 1.

Increase in IL-1β levels in the APP/PS1 KI mouse as a function of animal age, and selective attenuation of IL-1β by extended administration of MW-151 in a chronic intervention paradigm. A, Neocortical tissue from WT (open circles) or APP/PS1 mice (closed circles) was dissected from the brains of mice at different ages, and levels of IL-1β mRNA and protein were determined by qPCR or MSD ELISA, respectively. B, APP/PS1 mice were treated in a chronic intervention paradigm with either saline vehicle (veh; open bars) or MW-151 (closed bars; 2.5 mg/kg i.p. three times weekly) beginning at early age (6 months old). At the end of the treatment period, when mice were 11 months old, neocortical tissue was analyzed by MSD ELISA for the levels of IL-1β and IL-10. MW-151 selectively attenuated the proinflammatory cytokine IL-1β, but not the anti-inflammatory cytokine IL-10. *p < 0.05 compared to WT mice at 11 months old.

Figure 2.

Chronic MW-151 administration attenuates microglial activation. A, Representative images of IBA1-stained microglia in cortex of 11-month old APP/PS1 mouse, demonstrating the positive pixel and nuclear algorithms used for quantification of the staining. A computer-generated markup of the algorithm is created by the ScanScope software, which validates the specificity of the method. In the positive pixel algorithm, the microglia shown in shades of gray (indicating intensity of positive staining) is segregated from the background shown as black. The markup in the nuclear algorithm shows the reliable detection and quantification of individual cells (shown by the black markup). B, C, The APP/PS1 mice treated with vehicle (veh; gray bars) show increased microglial staining compared to WT mice treated with vehicle (white bars). MW-151 treatment of the APP/PS1 mice (black bars) reduced the microglial activation as assessed by either the stained area (B) or the number of stained microglia (C). *p < 0.05 compared to vehicle-treated APP/PS1 mice. **p < 0.01 compared to vehicle-treated APP/PS1 mice.

Figure 3.

Chronic MW-151 administration attenuates astrocyte activation. A, Representative images of GFAP-stained sections. B, The APP/PS1 mice treated with vehicle (veh; gray bars) show increased astrocyte staining in cortex compared to WT mice treated with vehicle (white bars). MW-151 treatment of the APP/PS1 mice (black bars) reduced the astrocyte activation. C, MW-151 treatment had no effect on GFAP mRNA levels but did reduce mRNA levels of two other astrocyte markers, S100B and vimentin. **p < 0.01 compared to vehicle-treated APP/PS1 mice.

Figure 4.

Chronic MW-151 administration prevents synaptic loss. Neocortex tissue was analyzed for the levels of synaptic proteins by Western blots. Compared to WT mice treated with vehicle (veh; white bars), the APP/PS1 mice treated with vehicle (gray bars) show reduced levels of the synaptic proteins PSD95 (A), synaptophysin (B), syntaxin (C), and SNAP25 (D). MW-151 treatment of the APP/PS1 mice protected against this loss of synaptic protein levels, restoring the levels back toward WT values. *p < 0.05 compared to vehicle-treated APP/PS1 mice.

Figure 5.

Effects of short-term MW-151 treatment in acute intervention paradigm. Eleven-month-old mice were treated with either saline vehicle (veh; white bars, WT; gray bars, APP/PS1) or with 2.5 mg/kg/d MW-151 i.p. (black bars, APP/PS) once daily for 1 week. Neocortex tissue was harvested and endpoints were measured. A, Short-term MW-151 treatment reduced the levels of IL-1β, but not IL-10, and had no effect on microglial (IBA1) or astrocyte (GFAP) activation. B, MW-151 treatment prevented the loss of PSD95 and synaptophysin, but the protection against syntaxin and SNAP25 loss did not reach significance. *p < 0.05 compared to vehicle-treated APP/PS1 mice.

Figure 6.

No effect of MW-151 treatment on Aβ pathology. A, Representative images of Aβ-stained hemi-brain sections. B, C, In either the chronic intervention paradigm (B) or the acute intervention paradigm (C), MW-151 treatment of APP/PS1 mice did not reduce the amyloid plaque load when measured as a percentage of the area stained or as the number of plaques per unit area. There were also no differences in the number of small (20–300), medium (301–1000), or large (1001-∞) amyloid plaques detected. MW-151 treatment of APP/PS1 mice also had no significant effect on the levels of soluble or aggregated forms of Aβ, as measured by levels of Aβ40 or Aβ42 in PBS-soluble and FA-soluble fractions. veh, Vehicle.

Figure 7.

MW-151 increases LTP in the hippocampus of 12-month-old APP/PS1 mice. A, Representative CA3-CA1 synaptic responses from vehicle (veh)-treated and 30 μm MW-151-treated hippocampal slices from APP/PS1 mice, collected immediately before (dashed line) and 60 min after (solid line) LTP induction. B, Time plot of EPSP slope measures taken before and after 100 Hz stimulation in slices from WT mice (white circles) and APP/PS1 mice treated with (black circles) or without (gray circles) MW-151. C, Bar graph shows LTP levels across treatment conditions. Number symbol (#) indicates a significant decrease (p < 0.05) in LTP in vehicle-treated slices from APP/PS1 mice compared to vehicle-treated slices from WT mice. Asterisk indicates a significant increase (p < 0.05) in LTP in MW-151-treated slices from APP/PS1 mice relative to the APP/PS1 vehicle group. D, EPSP slope measures plotted against FV measures in each treatment condition. No effects of genotype or MW-151 treatment on synaptic strength were observed.