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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Neurosci. Author manuscript; available in PMC 2013 January 25.
Published in final edited form as:
PMCID: PMC3419360

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


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 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.

Keywords: neuroinflammation, synaptic damage, LTP, glia, interleukin


Alzheimer’s disease (AD) and related neurodegenerative disorders have an unmet need for therapies that alter disease progression. Evidence suggests that disease progression involves uncontrolled glial activation and neuroinflammation that contribute to neuronal/synaptic dysfunction, which in turn drives a vicious cycle of further glial activation and neuronal damage (Mrak and Griffin, 2005). Dissecting this cycle to find points of potential therapeutic intervention is compounded by the complexity of this multifactorial disease, the multiple roles of glia in homeostasis and host defense, and the diversity of inflammatory and immune response mechanisms (Nelson et al., 2009, 2011; Huang and Mucke, 2012; Wyss-Coray and Rogers, 2012).

The diverse array of physiological functions that glia perform, which are context-dependent, complicates the interpretation of experimental investigations and clinical observations related to AD pathology. For example, astrocytes and microglia interact with neurons at the synapse to modulate synaptic function and plasticity (Eroglu and Barres, 2010; Tremblay et al., 2011), and are also vital for host defense mechanisms and response to stress (Ransohoff and Perry, 2009). Therefore, health and homeostasis, disease progression and susceptibility, and therapeutic interventions involve a delicate balance in which glial responses must be modulated within a dynamic range.

Related to disease progression, extended overproduction of proinflammatory cytokines such as IL-1β can be particularly detrimental to neuronal function (Shaftel et al., 2008; Hein et al., 2010; Kitazawa et al., 2011). The potential for amelioration of cytokine-induced neuronal dysfunction through therapeutic intervention was shown by Hu et al. (2007) with the development of a small molecule experimental therapeutic, MW01-2-151SRM (here termed MW-151). MW-151 is a selective suppressor of beta amyloid (Aβ)-induced glia proinflammatory cytokine production, with resultant attenuation of the cytokine-induced loss of synaptic marker proteins (e.g., synaptophysin and PSD-95) and cognitive deficits (Hu et al., 2007). A major unmet challenge in the field, however, is the question of therapeutic time window in progressive neurological disorders like AD. The therapeutic time window is a component of drug dosing, a fundamental aspect of the pharmacological basis of drug action, and is linked to the clinical principle of pathology progression. For example, an optimal time window for therapeutic intervention is generally during a peak activity change in the targeted pathway involved in pathology progression.

To address the issue of whether glial activation and proinflammatory cytokine increases might be an early stage pathology progression feature that could be modulated by drugs such as MW151 with resultant improvement in neurologic endpoints, we examined an APP/PS1 humanized knock-in mouse model that exhibits age-dependent AD pathology progression. We found that proinflammatory cytokines begin to increase early in the pathology progression, and that repeat dose intervention with MW151 starting during this period resulted in significantly improved outcomes. While improvements were also seen with later short-term intervention, the earlier and more prolonged intervention yielded more robust effects. Neither dosing paradigm had a detectable effect on amyloid plaque load or Aβ levels. The results indicate that selective inhibition of the increasing proinflammatory cytokine production early in disease progression is beneficial at maintaining synaptic function.


Synthesis of MW-151

MW01-2-151SRM (2-(4-(4-methyl-6-phenylpyridazin-3-yl)piperazin-1-yl)pyrimidine) was synthesized and characterized as previously reported (Hu et al., 2007). The active pharmaceutical used for biological studies was the dihydrochloride hydrate for of the water-soluble, acidic drug. Low doses per administration were employed as noted, and are similar to those used previously for efficacy in diverse animal models of CNS diseases that involve excessive proinflammatory cytokine production as part of the pathology progression mechanism, including a model of early AD (Hu et al., 2007), TBI (Lloyd et al., 2008; Chrzaszcz et al., 2010), multiple sclerosis (Karpus et al., 2008), and seizures (Somera-Molina et al., 2007, 2009) at low doses similar to that used in this study.

APP/PS1 knock-in mice

The AD mouse model used is the APPNLh/NLh x PS1P264L/P264L mutant mice originally developed at Cephalon (Flood et al., 2002). This double mutant mouse was generated by using gene targeting knock-in (KI) technology to introduce the Swedish FAD K670N/M671L mutations and humanize the mouse Aβ sequence (NLh), and to introduce the proline to leucine (P264L) mutation in the mouse PS-1 gene (Reaume et al., 1996; Siman et al., 2000). Expression of both genes is driven by the endogenous promoters, and thus, this model shows AD pathology without APP or PS1 overproduction. The APP/PS1 KI mice were maintained on a CD-1/129 background, and wild-type (WT) mice were obtained from heterozygous APP/PS1 matings and maintained as a separate line for more than 20 generations of inbreeding, for use as controls. Mice were monitored for maintenance of the appropriate genotypes by PCR analysis of tail snip DNA (Anantharaman et al., 2006). Mice were maintained under 12h light/dark cycles and provided food and water ad libitum. All animal protocols followed the principles of animal care and experimentation in the Guide for the Care and Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use Committee of the University of Kentucky.

Treatment of mice

MW-151 was dissolved in 0.9% sterile NaCl (saline: Hospira NDC 0409-4888-10). Two experimental paradigms were used to test MW-151 efficacy in the APP/PS1 mice. In the extended administration prevention study (chronic intervention paradigm), MW-151 was administered at a low dose (2.5 mg/kg) by intraperitoneal (i.p.) injection three times per week (M,W,F), beginning when mice were 6 months old and ending at 11 months. In the short-term treatment study (acute intervention paradigm), MW-151 (2.5 mg/kg) was administered i.p. to 11 month old mice once daily for one week. Controls included APP/PS1 KI or WT mice administered vehicle control (saline). To conserve on animal numbers, WT mice were not administered MW-151. Each experimental group (WT + vehicle, APP/PS1 KI + vehicle, APP/PS1 KI + MW-151) started with 12 mice of either sex, and the numbers of males and females in each group were matched as closely as possible.

Euthanasia and brain tissue harvesting

Mice were euthanized with an overdose of sodium pentobarbital, and transcardially perfused with ice-cold phosphate buffered saline (PBS) for 5 min. The brains were rapidly removed and bisected along the sagittal plane. The right hemisphere was further dissected and flash frozen in liquid nitrogen and stored at −80°C for subsequent biochemical evaluation. The left hemisphere was immersion fixed in 4% paraformaldehyde overnight, prior to cryoprotection in a 30% sucrose/PBS solution.

Biochemical endpoint analyses

Neocortical tissue was homogenized using an Omni TH homogenizer, in a lysis buffer (1:10 w/v) containing PBS, 1 mM EDTA, and protease inhibitors (1 mM PMSF, 1 μg/ml leupeptin). Extracts were centrifuged at 12,000 × g for 20 min at 4°C in a Beckman Microfuge 18, and supernatants were used as the PBS soluble fraction. Pellets were suspended and re-homogenized in a detergent containing lysis buffer (tissue protein extraction reagent with Halt protease and phosphatase inhibitor cocktail: Thermo Scientific) at the same volume used for the first homogenization. Extracts were centrifuged at 12,000 × g for 20 min at 4°C, and supernatants used for measurement of synaptic proteins in the detergent soluble fraction. The pellet was then re-suspended and re-homogenized in 70% formic acid (FA). The FA homogenate was centrifuged at 1,000,000 × g at 4°C for 60 min. The supernatant was neutralized 1:20 with 1 M Tris (pH11) and was used as the FA soluble fraction. Levels of Aβ40 and Aβ42 were measured by ELISA assay using kits from Meso Scale Discovery (MSD) according to the manufacturer’s instructions. Levels of cytokines (IL-1β and IL-10) were measured by ELISA assay using kits from MSD according to the manufacturer’s instructions with minor modifications. Briefly, 50μl of the PBS soluble fraction was loaded per well of the MSD plate (100–200μg of protein). The sample was incubated in the plate overnight at 4°C. All incubation steps were done using an Eppendorf MixMate at 1000 rpm. Cytokine levels were normalized to the total amount of protein in the sample loaded as determined by BCA Protein Assay (Thermo Scientific). The detection limits of the MSD assays are 1.5 pg/ml for IL-1β and 42 pg/ml for IL-10 in serum/plasma.

Levels of synaptic proteins were measured by Western blots as previously described (Xing et al., 2011). Briefly, 1μg of the detergent-solubilized fraction was prepared in SDS buffer (LI-COR). The sample was separated on a 10% NuPAGE Novex Bis-Tris Midi Gel (Invitrogen). Proteins were transferred to nitrocellulose membrane using a dry blotting system (iBlot® Invitrogen). Blots were probed using reagents and manufacturer recommendations for Odyssey Infrared imaging system (LI-COR Biosciences). Blots were probed for the following primary antibodies: rabbit anti-PSD95 (1:2,000, Cell Signaling, Cat no. 3450); mouse anti-synaptophysin (clone SY38) (1:30,000, Millipore, Cat no. MAB5258); rabbit anti-syntaxin 1 (1:100,000, Millipore, Cat no. ab5820), mouse anti-SNAP 25 (clone 20) (1:10,000, BD Biosciences, Cat no. 610366). Rabbit anti-GAPDH (clone 14C10) (1:10,000, Cell Signaling, Cat no. 2118) was used as a loading control. Blots were visualized and analyzed on the Odyssey Infrared imaging system (LI-COR Biosciences), and integrated intensity values were used in statistics.

For gene expression, dissected neocortical tissues stored at −80 °C were used for RNA isolation using RNeasy mini-columns (Qiagen, Cat no. 74104) with on-column DNase treatment (Qiagen, Cat no. 79254) according to the manufacturer’s protocol. RNA quantity and quality were determined using A260/A280 readings by NanoDrop (Thermo Scientific). Reverse transcription (RT) was done following the manufacturer’s protocol using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Cat no. 4368814). A no template and a no RT control were conducted to control for contamination. Real-time PCR was performed using the TaqMan Gene Expression assay kit (Applied Biosystems, Cat no. 4444964) according to the manufacturer’s instructions on a ViiA 7 Real-Time PCR System (Applied Biosystems). The following TaqMan probes (Applied Biosystems) were used: GFAP (Mm00546086_m1), S100B (Mm00485897_m1), vimentin (Mm01333430_m1), IL-1β (Mm00434228_m1), and (table 1). Relative gene expression was calculated by the 2−ΔΔCT method.

Table 1
Selective effects of MW-151 on inflammation-related gene expression markers

Immunohistochemical staining

Coronal sections (30 μm) of the left hemisphere were made using a sliding microtome and a freezing stage, and stored in cryoprotectant at −20°C. Staining procedures were conducted on free-floating sections using every twelfth section of the entire left cerebral cortex. Primary and secondary antibodies were diluted in 3% normal goat serum (NGS: LAMPIRE Biological Laboratories, Cat no. 7332500) with 0.2% Triton X-100. Amyloid plaque burden was detected by rabbit polyclonal anti-Aβ N-terminal antibody (1:10,000, Invitrogen, Cat no. 44338100). Sections were pretreated with 70% formic acid, endogenous peroxidase activity was quenched with 3% H2O2 in methanol, prior to the tissue blocking in 10% NGS with 0.2% Triton X-100. The biotinylated secondary antibody (1:500, Jackson ImmunoResearch, Cat no. 111-065-144) was amplified using avidin-biotin substrate (ABC solution, Vector Cat no. PK-6100) followed by color development in 3,3′-Diaminobenzidine tetra-hydrochloride (DAB: Sigma, Cat no. D5637). Glial activation was detected with the following antibodies: rabbit anti-GFAP (1:10,000 Dako, Cat no. Z0334) for astrocytes, and rabbit anti-IBA1 (1:10,000, Wako, Cat no. 019-19741) for microglia. Endogenous peroxidase activity was quenched with 3% H2O2 in methanol, followed by blocking in 10% NGS with 0.2% Triton X-100. The horseradish peroxidase secondary antibodies (1:500, Jackson ImmunoResearch, Cat no. 111-035-003) were visualized with DAB.

Quantitative image analysis of immunohistochemical stains

The Aperio ScanScope XT digital slide scanner was used to image the entire stained slide at 20x magnification to create a single high-resolution digital image. The neocortex and hippocampus were outlined using the Aperio Image Scope software. The Aperio positive pixel count algorithm (version 9) was used to quantify the amount of specific staining in the region. The number of positive pixels was normalized to the number of total pixels (positive and negative) to account for variations in the size of the region sampled. The Aperio nuclear algorithm (version 9) was used to quantify the number of cells or the number and size of Aβ plaques in the outlined region. To quantify the number of plaques as a function of size, a threshold was set in the nuclear algorithm so only plaques that were contained in the size bin would be quantified. Color and intensity thresholds were established to detect the immunostaining as positive pixels and background staining as negative pixels. Once conditions were established for an immunohistochemical stain, the entire batch of slides was analyzed with the same parameters. The resulting color markup of the analysis was confirmed for each slide. All quantifications were done by a blinded observer.

Slice Electrophysiology

Methods for preparing acute brain slices for assessment of CA1 synaptic function and plasticity were similar to our previous methods (Norris and Scheff, 2009; Mathis et al., 2011). Briefly, twelve-month-old WT and APP/PS1 KI mice were decapitated under CO2 anesthesia and brains rapidly removed and stored in ice-cold artificial cerebrospinal fluid (ACSF) containing: 124mM NaCl, 2mM KCl, 1.25mM KH2PO4, 2mM MgSO4, 26mM NaHCO3, 10mM dextrose, saturated with 95% O2 and 5% CO2. Cerebral hemispheres were blocked, mounted, and sectioned (400μm) in ice-cold Ca2+-free ACSF with a vibrating microtome (Leica Biosystems, Richmond, IL). Slices were then stored on nets in a humidified interface holding chamber and bathed in CaCl2 (2mM)-containing ACSF (32°C) with saline vehicle or 30μM MW-151. After a 1.5–2 h incubation period, slices were transferred to a modified RC-22 chamber (Warner Instruments, Hamden, CT) affixed to the stage of a Nikon E600 microscope (Nikon Instruments Inc., Melville, NY) and perfused (1–2 mL/min) with oxygenated CaCl2 (2mM)-containing ACSF (32°C). Synaptic responses were evoked by electrical stimulation of CA3 Schaffer collaterals and recorded in CA1 stratum radiatum as described previously (Norris and Scheff, 2009; Mathis et al. 2011). To generate synaptic strength curves, excitatory postsynaptic potential slope (EPSP) amplitudes were measured at nine stimulus intensity levels (30, 50, 100, 150, 200, 250, 300, 400, and 500 mA) and plotted against corresponding fiber volley (FV) amplitudes. For some slices, stimulus intensity was reset after the synaptic strength curve to evoke a 1 mV EPSP every 30 sec for at least 20 min prior to the delivery of two 1 sec duration trains of 100 Hz stimulation (10 sec intertrain interval) for induction of long term potentiation (LTP). Synaptic responses were then collected for an additional 60 min. All electrophysiological recordings were conducted and analyzed by personnel who were blind to genotype and drug treatment conditions.


Statistical analysis was conducted using GraphPad prism software version 5 (GraphPad Software, San Diego California USA). Values are expressed as mean ± SEM. Groups of 2 were compared by unpaired T-test. Groups of 3 or more were compared by One-way analysis of variance (ANOVA), followed by Bonferroni Multiple Comparison Test. Significance was defined as p < 0.05. Synaptic strength curves were fit with a three parameter (i.e. max EPSP amplitude, half-maximal FV amplitude, and curve slope) sigmoidal function and compared across treatment groups using Z tests (Z values ≥ |2| were considered statistically significant). For each slice, LTP amplitudes were averaged 50–60 min after delivery of 100 Hz stimulation and normalized to the pre-100 Hz baseline. Significant effects of drug treatment on LTP were determined within genotype using paired t tests. Differences in LTP amplitude between WT and APP/PS1 KI mice were compared using an unpaired t test.


Validation of mechanism of action endpoint in APPxPS1 mice

We previously reported (Hu et al., 2007) that MW-151 is efficacious in a mouse model of AD pathology that involves infusion of human oligomeric Aβ1-42 and represents disease in its early stages. However, the effect of the compound in an AD model that exhibits more severe amyloid plaque pathology and age-dependent disease progression had not been examined. The first necessary step was to choose an AD mouse model that exhibits appropriate changes in the process that is being targeted by our small molecule compound. Since the mechanism of action of MW-151 is to attenuate cytokine overproduction from activated glia, it was important to select an animal model that exhibits increases in glial activation and cytokine production during the pathology progression. We selected the APPNLh/NLh x PS1P264L/P264L mutant mouse model (APP/PS1) originally developed at Cephalon (Flood et al., 2002), where gene expression in this humanized APP/PS1 double knock-in model is driven by endogenous promoters of the APP and PS-1 genes. This model preserves physiologically relevant APP expression levels, and demonstrates progressive AD pathology without APP overproduction. Most importantly, we verified that this model shows an age-dependent increase in levels of proinflammatory cytokines above that of the wild-type (WT) mice. As shown in Figure 1A, the levels of IL-1β mRNA and protein in the cortex increase with age in both the WT and APP/PS1 KI mice, with the KI mice showing a significantly higher level of IL-1β at 11 months compared to the WT mice. Thus, this is a relevant animal model in which to test in vivo efficacy of a cytokine response modulator.

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

Extended administration of MW-151 in chronic intervention paradigm reduces proinflammatory cytokine levels

Based on the time course of the cytokine rise, we designed a chronic dosing, prevention study paradigm where MW-151 (2.5 mg/kg i.p.) or saline vehicle was administered 3 times per week (M,W,F) beginning at 6 months (when cytokine levels are just beginning to increase) and ending at 11 months (when cytokine overproduction is easily apparent). The mice tolerated well the extended administration of MW-151, exhibiting no significant differences in survival or weight. As shown in Figure 1B, APP/PS1 KI mice administered MW-151 showed lower levels of brain IL-1β compared to vehicle-treated mice, but no decrease in the anti-inflammatory cytokine IL-10. These data confirm that MW-151 exhibits its mechanism of action, i.e. selective attenuation of brain proinflammatory cytokine levels, in this animal model.

Chronic MW-151 administration attenuates microglial and astrocyte activation

The effect of MW-151 treatment on glial activation was examined by IHC using anti-IBA1 antibody to label microglia and anti-GFAP antibody to label astrocytes. Immunostained sections were scanned and converted into a high-resolution digital image using the Aperio ScanScope digital slide scanner and the positive pixels algorithm or the nuclear algorithm to quantify the immunohistochemical staining. The Aperio ScanScope enables high-resolution image recognition and rigorous digital pathological quantitation. Figure 2A shows a representative field of IBA1-stained microglia in the cortex and digital images of the algorithms used to calculate microglial activation characteristics. MW-151 treatment of APP/PS1 KI mice reduced the microglial activation, when data were expressed as either the number of positive pixels (Figure 2B) or the number of stained cells/mm2 (Figure 2C).

Figure 2
Chronic MW-151 administration attenuates microglial activation

In addition to effects on microglia activation, MW-151 administration also attenuated astrocyte activation. Figure 3A illustrates the GFAP-labeled hemibrain sections. MW-151 treatment reduced the astrocyte activation in cortex (Figure 3B) and hippocampus (data not shown), as quantified from the IHC. Interestingly, MW-151 had no effect on GFAP mRNA levels (Figure 3C). MW-151 treatment did, however, reduce the mRNA levels of two other astrocyte markers. There was a significant decrease in S100B mRNA and a trend toward a decrease in vimentin mRNA (Figure 3C).

Figure 3
Chronic MW-151 administration attenuates astrocyte activation

Chronic MW-151 administration prevents synaptic protein loss

The ability of chronic administration of MW-151 to lead to beneficial neuronal changes was tested by measuring the levels of a panel of synaptic protein markers by Western blot analysis. The APP/PS1 KI model exhibits synaptic dysfunction as seen by a reduction in the levels of synaptic proteins compared to WT mice (Figure 4). MW-151 treatment of APP/PS1 KI mice prevented the loss of the synaptic proteins PSD95 (Figure 4A), synaptophysin (Figure 4B), syntaxin (Figure 4C) and SNAP25 (Figure 4D).

Figure 4
Chronic MW-151 administration prevents synaptic loss

Selectivity of MW-151

In the AD brain, microglia exhibit a complex and heterogeneous set of responses in attempts to restore homeostasis. Although our understanding of the consequences of particular microglia responses to the neurodegenerative process in AD is limited, the classical inflammatory activation state (termed M1) can lead to overproduction of proinflammatory cytokines and subsequent neuronal damage, while an alternative activation state (termed M2) may be involved in attempts to prevent further damage or promote tissue repair. To test the selectivity of MW-151 action in terms of its effects on selected microglia and neuron responses, we measured a panel of markers by qPCR in the cortex from the vehicle-treated or MW-151-treated APP/PS1 KI mice. The endpoints tested represent microglia markers, toll-like receptors, M1 and M2 endpoints, neuroimmune regulatory molecules, and a damage-associated molecular pattern (DAMP) molecule. Table 1 shows that MW-151 modulates some endpoints but not others. For example, TNFα is reduced, but NOS2 is not, even though both are considered M1 inflammatory molecules. Similarly, one of the M2 markers is changed (TGFβ) but not the other (IGF1). Other M2 markers in the qPCR array (fizz, Arg1, BDNF) could not be measured accurately because they were at very low to undetected levels in the assay. These results with M2 markers are consistent with our previous observations (data not shown) that in the absence of external stressor stimuli, this APP/PS1 KI mouse model does not express many of the standard M2 markers to an appreciable degree. Other microglia markers also showed selective responses, in that IBA1 and CD68 are reduced, but CD45 is not. The neuroimmune regulatory molecules CD200 and fractalkine are increased, and the DAMP HMGB1 is decreased by MW-151 treatment. Although the functional significance of changes in these specific responses was not explored as part of this study, these data confirm that MW-151 is selective in its action and not a pan-suppressor of microglia and neuron responses.

Short-term MW-151 treatment in acute intervention paradigm

To test whether MW-151 is efficacious in older mice where disease pathology is already present, we administered MW-151 (2.5 mg/kg i.p.) or saline vehicle to 11 month old mice once daily for one week. As shown in Figure 5A, short-term administration of MW-151 in the acute intervention treatment paradigm attenuated IL-1β production without significant reductions in IL-10, and had no effect on microglial (IBA1) or astrocyte (GFAP) activation. Analysis of synaptic protein levels (Figure 5B) showed that MW-151 significantly protected the brain against loss of PSD95 and synaptophysin, but was less effective against loss of syntaxin and SNAP25.

Figure 5
Effects of short-term MW-151 treatment in acute intervention paradigm

No effects of MW-151 on amyloid plaque load or Aβ40 or Aβ42

We examined the effect of MW-151 treatment on amyloid plaque burden in both the chronic and acute dosing paradigms, by staining brain sections with a polyclonal anti-Aβ N-terminal antibody. This antibody reacts with both Aβ40 and Aβ42 and is specific for human Aβ. As shown in Figure 6A, the 11 month old APP/PS1 mice exhibit abundant numbers of amyloid plaques at this age, similar to previous reports using this model (Flood et al., 2002; Anantharaman et al., 2006; Murphy et al., 2007). Analysis of amyloid plaque staining in the cortex demonstrates that MW-151 treatment in either the chronic intervention paradigm (Figure 6B) or the acute intervention paradigm (Figure 6C) had no significant effect on amyloid plaque burden. There was also no effect of MW-151 treatment on amyloid plaque load in the hippocampus (data not shown). The lack of effect was seen when the data were expressed either as % of the area stained with anti-Aβ antibody or as number of plaques per mm2. In addition, MW-151 treatment had no effect on the size of the amyloid plaques detected. To determine if there was any effect of MW-151 treatment on the levels of Aβ, we prepared PBS soluble, detergent soluble, and FA soluble fractions of cortex, and measured the levels of Aβ40 and Aβ42 by quantitative Aβ ELISA. No appreciable Aβ was detected in the detergent soluble fraction (data not shown). The amount of Aβ40 or Aβ42 in either the PBS soluble or FA soluble fractions was not significantly different in the APP/PS1 KI mice treated with vehicle or with MW-151, in either the chronic (Figure 6B) or acute (Figure 6C) intervention paradigm. These data are consistent with the lack of effect on amyloid plaque burden, and show that Aβ pathology overall was not significantly changed by MW-151 treatment.

Figure 6
No effect of MW-151 treatment on Aβ pathology

MW-151 treatment rescues impairments in LTP

LTP is a widely studied experimental model of synaptic plasticity implicated in learning and memory (Bliss and Collingridge, 1993; Malenka and Nicoll, 1999). Because of the well-established link between elevated proinflammatory cytokine levels and impairments in LTP, along with observations that anti-cytokine strategies rescue LTP deficits (Rowan et al., 2007; Lynch, 2010; Yirmiya and Goshen, 2011), we investigated the effect of MW-151 on synaptic function and plasticity by LTP measurements. Coronal brain slices from twelve-month-old APP/PS1 KI mice were prepared and incubated for ≥ 1.5 h in oxygenated ACSF containing saline vehicle or 30 μM MW-151. Electrically evoked EPSPs were obtained in hippocampal CA1 stratum radiatum, and LTP was induced (Figure 7A) using 100 Hz stimulus trains as described previously (Mathis et al., 2011; Norris and Scheff, 2009). Similar to previous findings with other APP/PS1 mouse models (e.g. Trinchese et al., 2004), slices from vehicle-treated APP/PS1 mice exhibited significantly reduced LTP compared to slices from age-matched WT controls (n = 5) (Figure 7B,C). In contrast, LTP in MW-151-treated slices from the APP/PS1 group was similar in magnitude to the WT group, and significantly greater than that shown by vehicle-treated APP/PS1 mice (p < 0.05). No genotype or drug treatment effects were observed for other synaptic function markers, including basal synaptic strength (Figure 7D) and paired-pulse facilitation (data not shown). The results suggest that MW-151 treatment selectively enhances synaptic plasticity mechanisms in APP/PS1 KI mice.

Figure 7
MW-151 increases LTP in the hippocampus of 12 month old APP/PS1 mice


We report here several findings with important implications for future glia-targeted AD therapeutic strategies. First, we show in an APP/PS1 KI mouse model of AD pathology that pharmacological intervention with a selective, small molecule inhibitor of glial proinflammatory cytokine overproduction is efficacious at attenuating microglial and astrocyte activation and proinflammatory cytokine levels, which prevents loss of synaptic proteins and impairments in synaptic plasticity (LTP). Second, there were no adverse effects observed with extended treatment of aging mice with a therapy that targets excessive proinflammatory cytokine production. Third, the beneficial effects of MW-151 occurred in the absence of a detectable change in amyloid plaque load or in levels of soluble or aggregated forms of Aβ40 or Aβ42 measured in PBS soluble and FA soluble neocortical fractions. Fourth, although MW-151 is effective when administration is started at either early or later stages of disease pathology progression, the compound is more effective in a preventative mode where administration is initiated before full later stage pathology is emerging. Overall, our results show that selective inhibition of the increasing glia proinflammatory cytokine response in early time windows of disease progression is beneficial at maintaining synaptic function, suggesting the importance of therapeutic time window considerations in future design of efficacy studies of neuroinflammation-targeted drugs.

The observation that MW-151 was more efficacious when administered in a preventative paradigm before onset of overt pathology implies that drugs that target neuroinflammation may be most beneficial when used early in the course of disease pathogenesis. This is consistent with many studies that demonstrate that glial activation and neuroinflammation are early events in AD pathogenesis (e.g., see Cagnin et al., 2001; Mrak and Griffin, 2005; Griffin and Barger, 2010; Carter et al., 2012); recent GWAS data that show that many of the genes associated with increased AD risk are inflammation-related (Sleegers et al., 2010; Guerreiro et al., 2012); and epidemiological and population-based clinical studies that suggest an association between early use of nonsteroidal anti-inflammatory drugs (NSAIDs) and reduced risk of developing AD (McGeer et al., 1996; in’t Veld et al., 2001; Zandi and Breitner, 2001).

In addition, analysis of data (Breitner et al., 2011; Leoutsakos et al., 2012) from the Alzheimer’s Disease Anti-inflammatory Prevention Trial (ADAPT) randomized clinical trial suggest that the treatment effects of NSAIDs differ depending on the stage of disease, with potential beneficial effects in asymptomatic individuals with little or no cognitive decline at the time of drug use, but harmful effects in later stages of AD pathogenesis. Although MW-151 is not an NSAID, the results reported here are consistent with the idea that suppression of CNS inflammation is likely to be effective as a prevention strategy, before the self-propagating, proinflammatory cytokine cycle is too far advanced during disease progression. However, MW-151 was also able to block cytokine production and prevent the decrease in some synaptic proteins even when administered at a later stage of pathology progression (to 11-month old mice for only a week). These initial observations will need to be followed up with more detailed studies on therapeutic window, but they suggest that future anti-inflammatory strategies aimed at selective fine tuning of neuroinflammation might be useful not only to prevent or delay onset of AD, but potentially as part of therapeutic strategies to treat existing disease, which would involve co-therapy with other drugs.

MW-151 was discovered using a functional in vivo screen (Ralay Ranaivo et al., 2006; Hu et al., 2007) that employed an animal model of injury induced by toxic aggregates of human Aβ1-42. That model lacked an age-dependent progressive pathology and did not have an amyloid plaque-rich environment. The APP/PS1 KI model used here exhibits progressive amyloid plaque deposition, beginning at around 9 months of age and increasing with age (Flood et al., 2002; Anantharaman et al., 2006; Murphy et al., 2007). This model also exhibits progressive pathological alterations in bidirectional synaptic plasticity, cognitive measures, synaptosomal membrane lipids, and oxidative stress markers as a function of animal development and maturation (Anantharaman et al., 2006; Chang et al., 2006; Bruce-Keller et al., 2011). We have shown here that the APP/PS1 KI model also demonstrates an early and progressive increase in brain proinflammatory cytokine levels, and that dampening of the proinflammatory cytokine production and the associated glial activation is beneficial to synaptic function. In this regard, it is interesting to note that these beneficial effects of MW-151 occurred in the absence of a detectable effect on amyloid plaque load or a change in PBS soluble or FA soluble Aβ40/42 levels, demonstrating that MW-151 treatment does not shift the levels of soluble toxic Aβ oligomers or insoluble fibrils.

There is inconsistency in the literature with respect to the effect of blocking cytokine signaling on Aβ pathology. Some studies have reported that interference with cytokine signaling by anti-inflammatory drug treatment or genetic deletion strategies results in reduced amyloid plaque load (Lim et al., 2000; Jantzen et al., 2002; Yan et al., 2003; Morihara et al., 2005; He et al., 2007; Yamamoto et al., 2007). However, other studies have shown that cytokine suppressing strategies can reduce brain inflammatory responses and attenuate markers of neuronal pathology, with little or no effect on amyloid plaque pathology (Ralay Ranaivo et al., 2006; Kitazawa et al., 2011). Therefore, it is possible that an impact on amyloid plaques might be due to the model being used, the pharmacodynamic profile of the particular drug, the therapeutic time window of treatment and pathology development, or any combination of these variables. Our results with MW-151 pharmacodynamics in this particular model clearly demonstrate that synaptic and glia endpoints, related respectively to efficacy and in vivo mechanism of drug action, can be modulated independent of effects on Aβ. The linkage to tau pathology or its interface with Aβ pathology (e.g., see Zilka et al., 2012) cannot be addressed in this model because the APP/PS1 KI mouse lacks AD-relevant tau pathology. Thus, the interplay between glia proinflammatory cytokine-linked neuropathologies and AD-relevant tau neurofibrillary pathology remains to be addressed in future studies.

The linkage between changes in proinflammatory cytokine levels and neurologic outcomes, revealed by the improvement in synaptic endpoints (synaptic protein levels and LTP) after attenuation of proinflammatory cytokine levels by MW-151 treatment, is consistent with previous reports of MW-151 efficacy in other rodent CNS injury models, including oligomeric Aβ-induced injury (Hu et al., 2007), kainic acid-induced seizures (Somera-Molina et al., 2007, 2009), TBI models (Lloyd et al., 2008; Chrzaszcz et al., 2010), and the EAE model of multiple sclerosis (Karpus et al., 2008). These previous studies documented that MW-151 efficacy at reducing injury-induced upregulation of proinflammatory cytokine levels was associated with a reduction in long-term neuronal injury and attenuation of hippocampal-dependent cognitive deficits. Our findings are also consistent with reports that have linked other anti-neuroinflammatory interventions with changes in synaptic plasticity. For example, pharmacological interventions and/or genetic manipulations that suppress proinflammatory cytokine signaling normalize synaptic function in primary neural cultures (Sama et al., 2008) and ameliorate LTP deficits associated with aging (Griffin et al., 2006; Cowley et al., 2012) and Aβ (Rowan et al., 2007; Kotilinek et al., 2008; Yirmiya et al., 2011).

Overall, the data reported here add to the growing body of evidence that had identified dysregulated glial activation and proinflammatory cytokine overproduction as a common early pathophysiologic mechanism and potential therapeutic target in diverse neurodegenerative disorders. In addition, the results extend this state of knowledge to therapeutic intervention possibilities, especially the importance of therapeutic time window considerations in design of intervention strategies.


We thank Edgardo Dimayuga, Saktimayee M. Roy, James Schavocky and Bin Xing for their assistance with various aspects of this work. This research was supported in part by funds from the American Health Assistance Foundation (LVE), an Alzheimer’s Association Zenith award (LVE), a gift from the Kleberg Foundation (CMN), and NIH grants P01 AG005119 (LVE, DSC), R01 AG027297 (CMN), R01 NS056051 (DMW), R01 AG031311 (DMW), and S10 RR026489. ADB is a postdoctoral fellow supported by NIH F32 AG037280.


Conflict of Interest: None


  • Anantharaman M, Tangpong J, Keller JN, Murphy MP, Markesbery WR, Kiningham KK, St Clair DK. β-amyloid mediated nitration of manganese superoxide dismutase. Implications for oxidative stress in a APPNLh/NLh x PS-1P264L/P264L double knock-in mouse model of Alzheimer’s disease. Amer J Pathol. 2006;168:1608–1618. [PubMed]
  • Bliss TVP, Collingridge GL. A synaptic model of memory: long-term potentiation in the hippocampus. Nature. 1993;361:31–39. [PubMed]
  • Breitner JC, Baker LD, Montine TJ, Meinert CL, Lyketsos CG, Ashe KH, Brandt J, Craft S, Evans DE, Green RC, Ismail MS, Martin BK, Mullan MJ, Sabbagh M, Tariot PN. for the ADAP research group. Extended results of the Alzheimer’s disease anti-inflammatory prevention trial. Alz & Dem. 2011;7:402–411. [PMC free article] [PubMed]
  • Bruce-Keller AJ, Gupta S, Knight AG, Beckett TL, McMulle JM, Davis PR, Murphy MP, Van Eldik LJ, St Clair D, Keller JN. Cognitive impairment in humanized APPxPS1 mice is linked to Aβ1-42 and NOX activation. Neurobiol Disease. 2011;44:317–326. [PMC free article] [PubMed]
  • Cameron B, Landreth GE. Inflammation, microglia and Alzheimer’s disease. Neurobiol Dis. 2010;37:503–509. [PMC free article] [PubMed]
  • Cagnin A, Brooks DJ, Kennedy AM, Gunn RN, Myers R, Turkheimer FE, Jones T, Banati RB. In-vivo measurement of activated microglia in dementia. Lanet. 2001;358:461–467. [PubMed]
  • Carter SF, Schöll M, Almkvist O, Wall A, Engler H, Långström B, Nordberg A. Evidence for astrocytosis in prodromal Alzheimer disease provided by 11C-deuterium-L-deprenyl: a multitracer PET paradigm combining 11C-Pittsburgh compound B and 18F-FDG. J Nucl Med. 2012;53:37–46. [PubMed]
  • Chang EH, Savage MJ, Flood DG, Thomas JM, Levy RB, Mahadomrongkul V, Shirao T, Aoki C, Huerta PT. AMPA receptor downscaling at the onset of Alzheimer’s disease pathology in double knockin mice. Proc Natl Acad Sci USA. 2006;103:3410–3415. [PubMed]
  • Chrzaszcz M, Venkatesan C, Dragisic T, Watterson DM, Wainwright MS. Minozac treatment prevents increased seizure susceptibility in a mouse “two-hit” model of closed skull traumatic brain injury and electroconvulsive shock-induced seizures. J Neurotrauma 2010. 2010;27:1283–1295. [PMC free article] [PubMed]
  • Cowley TR, O’Sullivan J, Blau C, Deighan BF, Jones R, Kerskens C, Richardson JC, Virley D, Upton N, Lynch MA. Rosiglitazone attenuates the age-related changes in astrocytosis and the deficit in LTP. Neurobiol Aging. 2012;33:162–175. [PubMed]
  • Craft JM, Watterson DM, Frautschy SA, Van Eldik LJ. Aminopyridazines inhibit β-amyloid induced glial activation and neuronal damage in vivo. Neurobiol Aging. 2004;25:1283–1292. [PubMed]
  • Craft JM, Van Eldik LJ, Zasadzki M, Hu W, Watterson DM. Aminopyridazines attenuate hippocampus dependent behavioral deficits induced by human Aβ in a murine model of neuroinflammation. J Mol Neurosci. 2004;24:115–122. [PubMed]
  • Eroglu C, Barres BA. Regulation of synaptic connectivity by glia. Nature. 2010;468:223–231. [PubMed]
  • Flood DG, Reaume AG, Dorfman KS, Lin Y-G, Lang DM, Trusko SP, Savage MJ, Annaert WG, De Strooper B, Siman R, Scott RW. FAD mutant PS-1 gene-targeted mice: increased Aβ42 and Aβ deposition without APP overproduction. Neurobiol Aging. 2002;23:335–348. [PubMed]
  • Griffin R, Nally R, Nolan Y, McCartney Y, Linden J, Lynch MA. The age-related attenuation in long-term potentiation is associated with microglial activation. J Neurochem. 2006;99:1263–1272. [PubMed]
  • Griffin WST, Barger SW. Neuroinflammatory cytokines – the common thread in Alzheimer’s pathogenesis. US Neurol. 2010;6:19–27. [PMC free article] [PubMed]
  • Guerreiro RJ, Gustafson DR, Hardy J. The genetic architecture of Alzheimer’s disease: beyond APP, PSENs and APOE. Neurobiol Aging. 2012;33:437–456. [PMC free article] [PubMed]
  • He P, Zhong Z, Lindholm K, Berning L, Lee W, Lemere C, Staufenbiel M, Li R, Shen Y. Deletion of tumor necrosis factor death receptor inhibits amyloid β generation and prevents learning and memory deficits in Alzheimer’s mice. J Cell Biol. 2007;178:829–841. [PMC free article] [PubMed]
  • Hein AM, Stasko MR, Matousek SB, Scott-McKean JJ, Maier SF, Olschowka JA, Costa ACS, O’Banion MK. Sustained hippocampal IL-1β overexpression impairs contextual and spatial memory in transgenic mice. Brain Behav Immun. 2010;24:243. [PMC free article] [PubMed]
  • Hu W, Ralay Ranaivo H, Roy SM, Behanna HA, Wing LK, Munoz L, Guo L, Van Eldik LJ, Watterson DM. Development of a novel therapeutic suppressor of brain proinflammatory cytokine up-regulation that attenuates synaptic dysfunction and behavioral deficits. Bioorg Med Chem Lett. 2007;17:414–418. [PMC free article] [PubMed]
  • Huang Y, Mucke L. Alzheimer mechanisms and therapeutic strategies. Cell. 2012;148:1204–1222. [PMC free article] [PubMed]
  • in’t Veld BA, Ruitenberg A, Hofman A, Launer LJ, Van Duijn CM, Stijnen T, Breteler MMB, Stricker BHC. Nonsteroidal anti-inflammatory drugs and the risk of Alzheimer’s disease. New Engl J Med. 2001;345:1515–1521. [PubMed]
  • Jantzen PT, Connor KE, DiCarlo G, Wenk GL, Wallace JL, Rojiani AM, Coppola D, Morgan D, Gordon MN. Microglial activation and β-amyloid deposit reduction caused by a nitric oxide-releasing nonsteroidal anti-inflammatory drug in amyloid precursor protein plus presenilin-1 transgenic mice. J Neurosci. 2002;22:2246–2254. [PubMed]
  • Karpus WJ, Reynolds N, Behanna HA, Van Eldik LJ, Watterson DM. Inhibition of experimental autoimmune encephalomyelitis by a novel small molecular weight pro-inflammatory cytokine suppressing drug. J Neuroimmunol. 2008;203:73–78. [PMC free article] [PubMed]
  • Kitazawa M, Cheng D, Tsukamoto MR, Koike MA, Wes PD, Vasilevko V, Cribbs DH, LaFerla FM. Blocking IL-1 signaling rescues cognition, attenuates tau pathology, and restores neuronal β-catenin pathway function in an Alzheimer’s disease model. J Immunol. 2011;187:6539–6549. [PubMed]
  • Kotilinek LA, Westerman MA, Wang Q, Panizzon K, Lim GP, Simonyi A, Lesne S, Falinska A, Younkin LH, Younkin SG, Rowan M, Cleary J, Wallis RA, Sun GY, Cole G, Frautschy S, Anwyl R, Ashe KH. Cyclooxygenase-2 inhibition improves amyloid-beta-mediated suppression of memory and synaptic plasticity. Brain. 2008;131:651–664. [PMC free article] [PubMed]
  • Leoutsakos J-MS, Muthen BO, Breitner JCS, Lyketsos CG. For the ADAP Research Team. Effects of non-steroidal anti-inflammatory drug treatments on cognitive decline vary by phase of pre-clinical Alzheimer disease: findings from the randomized controlled Alzheimer’s disease anti-inflammatory prevention trial. Int J Geriatr Psychiatry. 2012;27:364–374. [PMC free article] [PubMed]
  • Lim GP, Yang F, Chu T, Chen P, Beech W, Teter B, Tran T, Ubeda O, Ashe KH, Frautschy SA, Cole GM. Ibuprofen suppresses plaque pathology and inflammation in a mouse model for Alzheimer’s disease. J Neurosci. 2000;20:5709–5714. [PubMed]
  • Lloyd E, Somera-Molina KC, Van Eldik LJ, Watterson DM, Wainwright MS. Suppression of acute proinflammatory cytokine and chemokine upregulation by post-injury administration of a novel small molecule improves long-term neurology outcome in a mouse model of traumatic brain injury. J Neuroinfl. 2008;5:28. [PMC free article] [PubMed]
  • Lynch MA. Age-related neuroinflammatory changes negatively impact on neuronal function. Frontiers in Aging Neurosci. 2010;1:1–8. [PMC free article] [PubMed]
  • Malenka RC, Nicoll RA. Long-term potentiation – a decade of progress? Science. 1999;285:1870–1874. [PubMed]
  • Mathis DM, Furman JL, Norris CM. Preparation of acute hippocampal slices from rats and transgenic mice for the study of synaptic alterations during aging and amyloid pathology. J Vis Exp. 2011;23:2330. pii. [PubMed]
  • McGeer PL, Schulzer M, McGeer EG. Arthritis and anti-inflammatory agents as possible protective factors for Alzheimer’s disease: a review of 17 epidemiologic studies. Neurology. 1996;47:425–432. [PubMed]
  • Morihara T, Teter B, Yang F, Lim GP, Boudinot S, Boudinot FD, Frautschy SA, Cole GM. Ibuprofen suppresses interleukin-1β induction of pro-amyloidogenic α2-antichymotrypsin to ameliorate β-amyloid (Aβ) pathology in Alzheimer’s models. Neuropsychopharmacol. 2005;30:1111–1120. [PubMed]
  • Mrak RE, Griffin WST. Glia and their cytokines in progression of neurodegeneration. Neurobiol Aging. 2005;26:349–354. [PubMed]
  • Munoz L, Ralay Ranaivo H, Roy SM, Hu W, Craft JM, McNamara LK, Wing Chico L, Van Eldik L, Watterson DM. A novel p38α MAPK inhibitor suppresses brain proinflammatory cytokine up-regulation and attenuates synaptic dysfunction and behavioral deficits in an Alzheimer’s disease mouse model. J Neuroinflammation. 2007;4:21. [PMC free article] [PubMed]
  • Murphy MP, Beckett TL, Ding Q, Patel E, Markesbery WR, St Clair DK, LeVine H, Keller JN. Aβ solubility and deposition during AD progression and in APPxPS-1 knock-in mice. Neurobiol Dis. 2007;27:301–311. [PubMed]
  • Nelson PT, Braak H, Markesbery WR. Neuropathology and cognitive impairment in Alzheimer disease: a complex but coherent relationship. J Neuropathol Exp Neurol. 2009;68:1–14. [PMC free article] [PubMed]
  • Nelson PT, Head E, Schmitt FA, Davis PR, Neltner JH, Jicha GA, Abner EL, Smith CD, Van Eldik LJ, Kryscio RJ, Scheff SW. Alzheimer’s disease is not “brain aging”: neuropathological, genetic, and epidemiological human studies. Acta Neuropathol. 2011;121:571–587. [PMC free article] [PubMed]
  • Norris CM, Scheff SW. Recovery of afferent function and synaptic strength in hippocampal CA1 following traumatic brain injury. J Neurotrauma. 2009;26:2269–2278. [PMC free article] [PubMed]
  • Ralay Ranaivo H, Craft JM, Hu W, Guo L, Wing LK, Van Eldik LJ, Watterson DM. Glia as a therapeutic target: selective suppression of human amyloid-β-induced upregulation of brain proinflammatory cytokine production attenuates neurodegeneration. J Neurosci. 2006;26:662–670. [PubMed]
  • Ransohoff RM, Perry VH. Microglial physiology: unique stimuli, specialized responses. Ann Rev Immunol. 2009;27:119–145. [PubMed]
  • Reaume AG, Howland DS, Trusko SP, Savage MJ, Lang DM, Greenberg BD, Siman R, Scott RW. Enhanced amyloidogenic processing of the beta-amyloid precursor protein in gene-targeted mice bearing the Swedish familial Alzheimer’s disease mutations and a “humanized” Abeta sequence. J Biol Chem. 1996;271:23380–23388. [PubMed]
  • Rowan MJ, Klyubin I, Wang Q, Hu NW, Anwyl R. Synaptic memory mechanisms: Alzheimer’s disease amyloid b-peptide-induced dysfunction. Biochem Soc Trans. 2007;35:1219–1223. [PubMed]
  • Sama MA, Mathis DM, Furman JL, Artiushin IA, Mohmmad Abdul H, Kraner SD, Norris CM. Interleukin-1β-dependent signaling between glia and neurons depends critically on astrocytic calcineurin/NFAT activity. J Biol Chem. 2008;283:21953–21964. [PMC free article] [PubMed]
  • Shaftel SS, Griffin WST, O’Banion MK. The role of interleukin-1 in neuroinflammation and Alzheimer disease: an evolving perspective. J Neuroinfl. 2008;5:7. [PMC free article] [PubMed]
  • Siman R, Reaume AG, Savage MJ, Trusko S, Lin Y-G, Scott RW, Flood DG. Presenilin-1 P264L knock-in mutation: differential effects on Aβ production, amyloid deposition, and neuronal vulnerability. J Neurosci. 2000;20:8717–8726. [PubMed]
  • Sleegers K, Lambert J-C, Bertram L, Cruts M, Amouyel P, Van Broeckhoven C. The pursuit of susceptibility genes for Alzheimer’s disease: progress and prospects. Trends in Genetics. 2010;26:84–93. [PubMed]
  • Somera-Molena KC, Robin B, Somera CA, Anderson C, Koh S, Behanna HA, Van Eldik LJ, Watterson DM, Wainwright MS. Glial activation links early-life seizures and long-term neurologic dysfunction: evidence using a small molecule inhibitor of pro-inflammatory cytokine upregulation. Epilepsia. 2007;48:1785–1800. [PubMed]
  • Somera-Molina KC, Nair S, Van Eldik LJ, Watterson DM, Wainwright MS. Enhanced microglial activation and proinflammatory cytokine upregulation are linked to increased susceptibility to seizures and neurologic injury in a ‘two-hit’ seizure model. Brain Res. 2009;1282:162–172. [PMC free article] [PubMed]
  • Tremblay ME, Stevens B, Sierra A, Wake H, Bessis A, Nimmerjahn A. The role of microglia in the healthy brain. J Neurosci. 2011;31:16064–16069. [PubMed]
  • Trinchese F, Liu S, Battaglia F, Walter S, Mathews PM, Arancio O. Progressive age-related development of Alzheimer-like pathology in APP/PS1 mice. Ann Neurol. 2004;55:801–814. [PubMed]
  • Van Eldik LJ, Thompson WL, Ralay Ranaivo H, Behanna HA, Watterson DM. Glia proinflammatory cytokine upregulation as a therapeutic target for neurodegenerative diseases: function-based and target-based discovery approaches. Intl Rev Neurobiol. 2007;82:277–296. [PubMed]
  • Wing LK, Behanna HA, Van Eldik LJ, Watterson DM, Ralay Ranaivo H. De novo and molecular target-independent discovery of orally bioavailable lead compounds for neurological disorders. Curr Alz Res. 2006;3:205–214. [PubMed]
  • Wyss-Coray T, Rogers J. Inflammation in Alzheimer disease – a brief review of the basic science and clinical literature. Cold Spring Harb Perspect Med. 2012;2:a006346. [PMC free article] [PubMed]
  • Xing B, Bachstetter AD, Van Eldik LJ. Microglia p38α MAPK is critical for LPS-induced neuron degeneration through a mechanism involving TNFα Mol Neurodegeneration. 2011;6:84. [PMC free article] [PubMed]
  • Yamamoto M, Kiyota T, Horiba M, Buescher JL, Walsh SM, Gendelman HE, Ikezu T. Interferon-γ and tumor necrosis factor-α regulate amyloid-β plaque deposition and β-secretase expression in Swedish mutant APP transgenic mice. Am J Pathol. 2007;170:680–692. [PubMed]
  • Yan Q, Zhang J, Liu H, Babu-Khan S, Vassar R, Biere AL, Citron M, Landreth G. Anti-inflammatory drug therapy alters β-amyloid processing and deposition in an animal model of Alzheimer’s disease. J Neurosci. 2003;23:7504–7509. [PubMed]
  • Yirmiya R, Goshen I. Immune modulation of learning, memory, neural plasticity and neurogenesis. Brain Behav Immun. 2011;25:181–213. [PubMed]
  • Zandi PP, Breitner JCS. Do NSAIDs prevent Alzheimer’s disease? And, if so, why? The epidemiological evidence. Neurobiol Aging. 2001;22:811–817. [PubMed]
  • Zilka N, Kazmerova Z, Jadhav S, Neradil P, Madari A, Obetkova D, Bugos O, Novak M. Who fans the flames of Alzheimer’s disease brains? Misfolded tau on the crossroad of neurodegenerative and inflammatory pathways. J Neuroinfl. 2012;9:47. [PMC free article] [PubMed]