SEARCH

SEARCH BY CITATION

Keywords:

  • Amyloid-β (Aβ);
  • interleukin-1β (IL-1β);
  • interleukin-4 (IL-4);
  • Long-term potentiation (LTP);
  • microglial activation;
  • minocycline

Abstract

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

It has been shown that Aβ inhibits long-term potentiation (LTP) in the rat hippocampus and this is accompanied by an increase in hippocampal concentration of IL-1β. Aβ also increases microglial activation, which is the likely cell source of IL-1β. Because IL-4 attenuates the effects of IL-1β in hippocampus, and microglial activation is inhibited by minocycline, we assessed the ability of both IL-4 and minocycline to modulate the effects of Aβ on LTP and IL-1β concentration. Following treatment with Aβ, IL-4 or minocycline, rats were assessed for their ability to sustain LTP in perforant path-granule cell synapses. We report that the Aβ-induced inhibition of LTP was associated with increases in expression of MHCII, JNK phosphorylation and IL-1β concentration, and that these changes were attenuated by treatment of rats with IL-4 and minocycline. We also report that Aβ-induced increases in expression of MHCII and IL-1β were similarly attenuated by IL-4 and minocycline in glial cultures prepared from neonatal rats. These data suggest that glial cell activation and the consequent increase in IL-1β concentration mediate the inhibitory effect of Aβ on LTP and indicate that IL-4, by down-regulating glial cell activation, antagonizes the effects of Aβ.

Abbreviations used
LTP

long-term potentiation

EPA

eicosapentaenoic acid

LPS

lipopolysaccharide

Evidence from previous studies has shown that injection of amyloid-β (Aβ) peptides into the brain inhibits long-term potentiation (LTP) in vivo (Walsh et al. 2002; Freir et al. 2003; Minogue et al. 2003; Klyubin et al. 2004) and in vitro (Saleshando and O’Connor 2000; Chen et al. 2002; Wang et al. 2002, 2004b; Freir et al. 2003). The evidence suggests that oligomeric Aβ exerts the most profound effect on synaptic plasticity (Walsh et al. 2002; Cleary et al. 2005; Lesne et al. 2006) although specific forms of fibrillar Aβ have been shown to inhibit LTP (Rowan et al. 2004).

The use of a variety of pharmacological agents has identified possible mechanisms by which Aβ-induced inhibition of LTP occurs. Thus inhibitors of inducible nitric oxide synthase as well as scavengers of superoxide reversed the Aβ-induced inhibition of LTP in vivo (Wang et al. 2004a). Inhibition of JNK activation has also been shown to attenuate the Aβ-induced inhibition of LTP in vivo (Minogue et al. 2003). All these inhibitors target inflammation emphasizing an important role for inflammatory changes in triggering the effects of Aβ. This is consistent with the data indicating that patients undergoing anti-inflammatory treatments for other reasons exhibited a reduced risk of developing AD (McGeer et al. 1996). The involvement of IL-1β in the pathogenesis of AD is supported by the observation that IL-1β expression is increased in CSF and in different brain areas of AD patients (Griffin et al. 1989; Blum-Degen et al. 1995; Sheng et al. 2000).

It has been reported that the inhibitory effect of intracerebroventricularly-injected Aβ on LTP in perforant path-granule cell synapses in vivo is mediated by IL-1β-induced activation of JNK (Minogue et al. 2003); this role of IL-1β is consistent with previous observations which indicated that increased hippocampal IL-1β concentration, and the accompanying increase in JNK activation, such as that associated with age or lipopolysaccharide (LPS) treatment, results in inhibition of LTP. Furthermore, eicosapentaenoic acid (EPA) treatment, which reversed the age-related and LPS-induced increase in IL-1β, such as that associated with age or lipopolysaccharide (LPS) treatment, resulted in restoration of LTP (Martin et al. 2002; Lynch et al. 2003).

Recent evidence has indicated that treatment of rats with IL-4, which down-regulates IL-1β and consequently attenuates IL-1β-induced signaling, also leads to restoration of LTP in aged rats and in rats treated with IL-1β or LPS (Barry et al. 2005; Nolan et al. 2005). The importance of IL-4 in modulating LTP has been highlighted by our most recent findings which indicate that EPA increases IL-4 expression in the hippocampus of aged rats and that this is likely to be the mechanism of action of EPA (Lynch et al. 2005).

Minocycline is a second-generation tetracycline and is known to have anti-inflammatory effects independent of their anti-microbial activity (Yrjanheikki et al. 1998); it inhibits microglial activation (Ryu et al. 2004; Griffin et al. 2006) and attenuates the age-related increase in IL-1β and deficit in LTP (Griffin et al. 2006). It has also been shown to decrease the effect of intracerebral injection of LPS in neonatal rats (Fan et al. 2005), to attenuate the increase in production of inflammatory cytokines in models of pain facilitation (Ledeboer et al. 2005) and to reduce Aβ-associated microglial activation. More recently, it was shown that minocycline reduced microglial activation following Aβ deposition in transgenic mice which over-expressed APP (Seabrook et al. 2006).

Here we considered that if the inhibitory effect of Aβ on LTP was primarily caused by its ability to increase IL-1β as a consequence of microglial activation, then IL-4, because it decreases microglial activation and down-regulates IL-1β, would ameliorate the Aβ-induced effect. Similarly, if the primary effect of Aβ is to increase microglial activation, then it follows that minocycline should exert an effect similar to IL-4. The data presented show that both IL-4 and minocycline attenuate the effect of Aβ on hippocampal function, that minocycline increases hippocampal IL-4 concentration and suggest that the action of IL-4 is a consequence of its ability to decrease glial cell activation.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Animals

Male Wistar rats (3–4 months old) were used in these experiments. Animals had free access to food and water, were housed in groups of 3–6 in a controlled environment (temperature: 20–22°C; 12 : 12 h light/dark cycle) and were maintained under veterinary supervision for the duration of all experiments. Animals were divided randomly into four treatment groups and there were six per group unless otherwise stated. For the experiments in which glial cultures were prepared, we used 1-day-old rats. All experiments were carried out under a license from the Department of Health and Children (Ireland) and with ethical approval from Trinity College Dublin Ethical Committee.

Preparation of Aβ1–42

1–42 (BioSource International, Camarillo, CA, USA) was dissolved in HPLC grade water to provide a 5 mg/mL stock solution, diluted to 1 mg/mL using sterile PBS and allowed to aggregate for 48 h at 37°C according to the manufacturer’s instructions. The presence of fibrillar Aβ in the injected preparation was demonstrated by assessing the binding of thioflavin T (ThT) (10 μL; 100 μmol/L; Sigma, Gillingham, UK). ThT fluorescence (435 nm excitation, 485 nm emission) increased maximally by 209% over the 48 h incubation period. The Aβ preparation was also assessed for oligomeric forms by gel electrophoresis and Coomassie blue staining. Briefly, samples (10 μL) were boiled for 2 min, loaded onto 15% SDS gels and the Aβ species were separated by application of a 32 mA current. Gels were rinsed in water, incubated overnight with the Coomassie G-250 solution (15 mL; GelCode Blue Stain Reagent, Pierce, Rockford, IL, USA) and photographed (Gel-Doc-It Bioimaging System, Ultraviolet Products Inc., Cambridge, UK). The predominant Aβ species was the 13.5 kDa species and therefore the preparation injected was therefore a mixture of fibrillar Aβ and oligomeric Aβ.

Treatment of rats and induction of LTP in vivo

Urethane-anesthetized rats were injected intracerebroventricularly (2.5 mm posterior and 0.5 mm lateral to Bregma) with Aβ1–42 (1 nmol/μL; 5 μL), IL-4 (20 μg/mL; 5 μL)) or both and 4 h later were assessed for their ability to sustain LTP in perforant path-granule cell synapses as previously described (Minogue et al. 2003). In a second series of experiments, rats were divided into four treatment groups; two groups of rats served as controls and two received minocycline. Minocycline (100 mg/kg body weight/day) was administered in the rats’ drinking water for 7 days. Assessment of water consumption indicated that there was an initial reduction of about 30% in intake in the minocycline-treated rats and further assessment of volume consumed indicated that the minocycline intake was between 60 and 71 mg/kg/day. After 7 days of treatment, control- and minocycline-treated rats were anaesthetized with urethane (1.5 mg/kg intraperitoneally) and injected intracerebroventricularly with Aβ (1 nmol/μL; 5 μL). Rats were placed in a stereotaxic frame, a bipolar stimulating electrode was positioned in the perforant path (4.4 mm lateral to lambda) and a unipolar recording electrode was positioned in the dorsal cell body region of the dentate gyrus (2.5 mm lateral and 3.9 mm posterior to Bregma). Test shocks were delivered at 30 s intervals, and after a stabilization period, responses were recorded for 10 min before, and 40 min after, tetanic stimulation (three trains of stimuli; 250 Hz for 200 ms; 30 s intertrain interval); this stimulation paradigm has been shown to induce saturable LTP in dentate gyrus in young urethane-anaesthetized rats (Lynch et al. 1985). Rats were killed by decapitation at the end of the period of electrophysiological recording and the brains were rapidly removed. The hippocampus was dissected free, one portion was flash frozen in liquid nitrogen and the remaining tissue was cross-chopped (350 × 350 μm) using a McIlwain tissue chopper, frozen in Krebs solution containing CaCl2 (2 mmol/L) and 10% DMSO as previously described (Martin et al. 2002), and stored at −80°C until required for analysis.

Preparation of primary glial cultures

Glial cultures were prepared from cortices of 1-day-old Wistar rats (Nolan et al. 2005). Dissected tissue was roughly chopped and added to pre-warmed Dulbecco’s modified Eagle medium (DMEM; Gibco BRL, Ireland) containing fetal bovine serum, penicillin (100 U/mL) and streptomycin (100 U/mL). Tissue was triturated, the suspension was filtered through a sterile mesh filter (40 μm), centrifuged at 2000 g for 3 min at 20°C and the pellet re-suspended in warmed DMEM. Re-suspended cells were plated in T10 flasks at a density of 0.25 × 106 cells/mL and incubated for 2 h before addition of warmed DMEM. Cells were grown at 37°C in a humidified 5% CO2: 95% air environment and media was changed every 3 days. Cultured cortical glia were incubated in the absence or presence of Aβ (2 μmol/L) or IFN-γ (50 ng/mL) with or without a 1 h pre-treatment with IL-4 (200 ng/mL). In a second series of experiments cells were incubated in control medium, medium with Aβ (2 μmol/L) alone or in the presence of anti-IFNγ antibody (1, 5, or 10 μg/mL; anti-rat antibody; R&D Systems, Abingdon, UK). In a third series of experiments cells were incubated in control medium or medium containing minocycline (30 μg/mL), Aβ (2 μmol/L) or both. In all cases supernatant was collected 24 h later and assessed for IL-1β concentration, and in the case of two of these experiments, cells were harvested for analysis of MHCII mRNA expression. In a final series of experiments, we assessed the role of JNK activation in the Aβ-induced release of IL-1β by pretreating cultured glia with the peptide inhibitor of JNK, D-JNKiI (2 μmol/L) for 4 h before adding Aβ (2 μmol/L). The mixed glial culture used in these studies consisted of approximately 30% microglia.

Expression of MHCII mRNA

MHCII mRNA expression was assessed by analyzing OX-6 mRNA in flash frozen samples prepared from hippocampus of control-, Aβ-, IL-4-, Aβ+IL-4- and minocycline-treated rats, or in harvested glia. cDNA synthesis was performed on 1 μg of total RNA using oligo (dT) primer (Superscript reverse transcriptase, Invitrogen, UK) at 1 unit/μg of RNA for 10 min at 65°C. Equal amounts of cDNA were used for PCR amplification for a total of 30 cycles. Primers were pre-tested through an increasing number of cycles to obtain reverse transcription-PCR products in the exponential range. The following sequences of primers were used: upstream, 5′-CAG TCA CAG AAG GCG TTT ATG-3′; downstream, 5′-TGC AGC ATC TGA CAG CAG GA-3′ and for rat β-actin mRNA expression; upstream, 5′-AGA AGA GCT ATG AGC TGC CTG AGG-3′; downstream, 5′-CTT CTG CAT CCT GTC AGC GAT GC-3′. The cycling conditions were as follows: 95°C for 300 s, 65°C for 60 s and 72°C for 120 s. The reaction was stopped by a final extension at 72° for 10 min and stored at 4°C. These primers generated OX-6 PCR products at 245 bp and β-actin PCR products at 250 bp. Equal volumes of PCR product from each sample were loaded onto 1.5% agarose gels and bands were separated by application of 90 V. Gels were photographed and quantified using densitometry. Estimation of mRNA expression was carried out using β-actin as a reference gene. No observable change in β-actin mRNA expression occurred in any of the treatment conditions.

Analysis of IL-1β, IFNγ, IL-6, TNFα and IL-4

IL-1β, IL-6, IFNγ and TNFα concentrations were analyzed in samples of homogenate prepared from hippocampus and in supernatant samples obtained from glial preparations using ELISA as previously described (Maher et al. 2005), (R&D Systems, BD Biosciences, San Jose, CA, USA); concentrations of IL-4 were only assessed in samples of hippocampal homogenate. Briefly, 96-well plates were coated with goat anti-rat IL-1β antibody (100 μL; 1 µg/mL in PBS containing 1% BSA, pH 7.3; R&D Systems), mouse anti-rat IL-4 antibody or IFNγ antibody (100 μL; 2 µg/mL in PBS, pH 7.3; R&D Systems), anti-rat IL-6 and anti-rat TNFα antibody (1 : 125 and 1 : 250, respectively in coating buffer, pH 9.5; BD Biosciences), incubated overnight, washed and incubated for 1 h with assay diluent (300 μL; PBS containing 1% BSA, and 0.05% NaN3, or PBS with 10% FBS for IL-6 and TNFα). After washing in PBS, triplicate samples and standards (100 µL; 0–1000 pg/mL, recombinant rat IL-1β or IL-4, or 0–2500 pg/mL recombinant rat IFNγ, IL-6 or TNFα) were added, incubation proceeded for 2 h and samples were washed and then incubated for 2 h in the presence of detection antibody (100 µL; 350 ng/mL in PBS containing 1% BSA, biotinylated goat anti-rat IL-1β; 150 ng/mL in PBS containing 1% BSA, biotinylated goat anti-rat IFNγ antibody; 50 ng/ml biotinylated goat anti-rat anti-IL-4, 1 : 125 dilution in assay diluent biotinylated anti-rat IL-6 and 1 : 250 dilution in assay diluent biotinylated anti-rat TNFα). Detection reagent (100 µL; HRP conjugated streptavidin; 1 : 200 dilution in PBS containing 1% BSA, or 1 : 250 dilution assay diluent for IL-4 and TNFα) was added, incubation continued for 20 min (or 2 h in the case of IL-4), samples were washed and substrate solution (100 µL; 1 : 1 mixture of H2O2 and tetramethylbenzidine) was added. Samples were incubated in the dark for 20 to 30 min and the reaction was stopped using 50 µl 1M H2SO4. Plates were read at 450 nm and cytokine concentrations were estimated from the appropriate standard curve and expressed as pg/mg protein.

Analysis of pJNK and JNK

Hippocampal tissue was homogenized in Krebs solution containing CaCl2 (2 mmol/L) and assessed for expression of pJNK1, pJNK2 and total JNK (tJNK) proteins by gel electrophoresis and immunoblotting. Tissue samples were equalized for protein concentration and diluted to a final protein concentration of 1 mg/mL. Aliquots (10 μL) were added to NuPAGE LDL sample buffer (Invitrogen, UK) containing NuPAGE reducing agent, heated at 70 °C for 10 min, and loaded onto 10% NuPAGE Novex Bis–Tris gels. Proteins were separated (200 V constant for 45 min) and transferred onto nitrocellulose membrane (30 V constant for 1 h). Membranes were blocked for 1 h in TBS 0.1% Tween 20 (TBS-T) and 5% BSA, and incubated overnight at 4°C with primary antibody (pJNK, 1 : 1000; tJNK, 1 : 1000; diluted in TBS-T with 2% BSA; Cell Signaling Technology Inc., Danvers, MA, USA) at 4°C. Membranes were washed three times in TBS-T, incubated with horseradish peroxidase-linked anti-rabbit antibody (1 : 1000 in TBS-T with 2% BSA) for 1 h, and washed again in TBS-T. Immunoreactive proteins were detected with enhanced chemiluminescence (Amersham Biosciences, Little Chalfont, UK). Values are expressed as the ratio of pJNK:tJNK.

Statistical analysis

A one-way analysis of variance was performed to determine whether there were significant differences between conditions. When this analysis indicated significance (at the 0.05 level), post hoc student Newmann–Keuls test analysis was used to determine which conditions were significantly different from each other.

Results

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We demonstrate that intracerebroventricular injection of Aβ significantly increased MHCII mRNA expression twofold in hippocampus (***p < 0.001: anova) and that this increase was completely abrogated in tissue prepared from animals that were treated with Aβ and IL-4 (+++p < 0.001: anova; Fig. 1a). In parallel with this, these data indicate significant Aβ-induced increases in IFNγ concentration (**p < 0.001; anova; Fig. 1b), and also IL-1β concentration (**p < 0.001: anova; Fig. 1c) consistent with the view that Aβ increases microglial activation. Intracerebroventricular injection of IL-4 prevented the Aβ-induced changes so that there was a statistically significant difference in MHCII mRNA expression as well as IFNγ and IL-1β concentration in tissue prepared from animals treated with Aβ alone and those treated with Aβ and IL-4 combined (+++p < 0.001: anova; Figs 1b and c). We also observed that injection of IL-4 led to a significant reduction in hippocampal IL-1β concentration (***p < 0.001; anova; Fig. 1c). Aβ also induced a significant increase (60%) in TNFα concentration (*p < 0.05; anova; Fig. 1d) and this was attenuated in tissue prepared from Aβ-treated rats which received IL-4 so that the mean value in this group (273.2 pg/mg ± 29.8 (SEM)) was not significantly different from the controls (249.9 pg/mg ± 22.9). Aβ induced an 89% increase in IL-6 and co-treatment with IL-4 attenuated this effect (Fig. 1e); therefore these changes mirrored those in TNFα but, in this case, did not reach statistical significance.

image

Figure 1.  Aβ-induced microglial activation is inhibited by IL-4 in vivo. (a) Mean MHCII mRNA expression, analyzed by using OX-6 RT-PCR was significantly increased in hippocampal tissue prepared from Aβ-treated (hatched bars, lanes 2 and 4 in the photograph), compared with control-treated (clear bars, lanes 1 and 3) rats (***p < 0.001; anova, n = 5). Mean MHCII mRNA expression was significantly decreased in tissue prepared from rats treated with Aβ and IL-4 (right-hand panels, lane 4) compared with those treated with Aβ alone (lane 3; +++p < 0.001; anova, n = 5). (b) The concentration of IFNγ was assessed in hippocampal homogenate by ELISA. Mean IFNγ concentration was significantly increased in hippocampal tissue prepared from Aβ-treated rats, compared with control-treated (**p < 0.01; anova, n = 6), while IL-4 reversed this increase (+++p < 0.001; anova, n = 6). (c) IL-1β concentration was significantly increased in hippocampal samples from Aβ-treated rats compared to control animals (**p < 0.01; anova). IL-4 significantly reduced IL-1β compared with control animals (***p < 0.001; anova). Co-treatment with Aβ and IL-4 significantly reduced IL-1β concentration relative to that induced by Aβ (+++p < 0.001; anova). (d) The concentration of TNFα was assessed in hippocampal homogenate by ELISA. Mean TNFα concentration was significantly increased in hippocampal tissue prepared from Aβ-treated rats, compared with control-treated (*p < 0.05; anova, n = 6), while IL-4 reversed this increase (n = 5). (e) IL-6 concentration was increased in hippocampal samples from Aβ-treated rats compared to control animals but was just outside statistical significance.

Download figure to PowerPoint

Analysis revealed a similar stimulatory effect of Aβ on microglia in vitro. These data indicate that both MHCII mRNA and IL-1β concentration were significantly increased (12- and 38-fold respectively) in Aβ-treated, compared with control-treated, glia (***p < 0.001 anova; in each case; Figs 2a and c, respectively) and that the effects of Aβ were completely abrogated by IL-4 so that mean values in preparations treated with Aβ plus IL-4 were significantly less than those in preparations treated with Aβ alone (+++p < 0.001; anova; Figs 2a and c, respectively). Similarly, MHCII mRNA and IL-1β concentration were significantly increased (12- and 42-fold, respectively) in IFNγ-treated, compared with control-treated glia (***p < 0.001 in each case; anova; Figs 2b and d, respectively) and the effects of IFNγ were completely abrogated by IL-4 so that mean values in preparations treated with IFNγ plus IL-4 were significantly less than those in preparations treated with Aβ alone (+++p < 0.001; anova; Figs 2b and d, respectively). Aβ also increased TNFα release from microglia (Control vs Aβ-treated glia; ***p < 0.001; anova) and IL-4 significantly reversed this Aβ-induced change (+++p < 0.001 compared with the value in Aβ-treated samples; anova; Fig. 2e); IL-4 exerted no significant effect on TNFα concentration. Significantly we found that the Aβ-induced increase in IL-1β concentration in glia (***p < 0.001; anova) was abrogated in a dose-dependent manner by co-treatment of cells with anti-IFNγ antibody; the data show that neither 1 nor 5 μg/mL antibody exerted any marked effect but 10 μg/ml significantly attenuated the Aβ-induced increase (+++p <  0.001; anova; Fig. 2f).

image

Figure 2.  Aβ-induced microglial activation is inhibited by IL-4 in vitro. Cultured cortical glia were incubated in the absence or presence of Aβ (2 μmol/L) or IFN-γ (50 ng/mL) with or without a 1 h pre-treatment with IL-4 (200 ng/mL). Cells were analyzed for expression of MHCII mRNA using OX-6 RT-PCR (a, b) and supernatant was removed and used for analysis of IL-1β and TNFα concentration (c, d, e). Representative photographs are presented for control (lane 1), Aβ (a) or IFNγ (b; lane 2), IL-4 (lane 3) and Aβ (a) or IFNγ (b) +IL-4 (lane 4) treatment groups. Aβ markedly increased MHCII mRNA expression (***p < 0.001; anova, n = 5) and IL-4 abrogated the Aβ-induced change (+++p < 0.001; anova). Similarly, IFNγ markedly increased MHCII mRNA expression (***p < 0.001; anova, n = 3) and IL-4 abrogated the IFNγ-induced change (+++p < 0.001; anova). Both Aβ and IFN-γ induced an increase in IL-1β (***p < 0.001; anova, n = 6). This was suppressed by pre-treating cells with IL-4 (+++p < 0.001; anova). (e) Mean TNFα concentration was also significantly increased following Aβ treatment, compared with control-treated cells (***p < 0.001; anova, n = 6), while IL-4 significantly reversed the increase (+++p < 0.001; anova, n = 5). (f) The significant Aβ-induced increase in IL-1β concentration in glia (***p < 0.001; anova) is dose-dependently decreased by co-treatment of cells with anti-IFNγ antibody and 10 μg/mL significantly attenuated the Aβ-induced increase (+++p < 0.001; anova; n = 3–6).

Download figure to PowerPoint

We assessed JNK activation in hippocampal tissue prepared from rats treated with Aβ and/or IL-4 and show that mean expression of phosphorylated JNK (pJNK) was significantly increased in tissue prepared from Aβ-treated rats; the data indicate that the increase was statistically significant in the case of both pJNK1 and pJNK2 (*p < 0.05 and **p < 0.01; anova: Figs 3a and b, respectively; 100% and 156% increases, respectively). These changes were attenuated in rats which also received IL-4 so that values in this group were significantly decreased compared with that in tissue prepared from rats which received Aβ alone (+p < 0.05 and ++p < 0.01; anova). The evidence suggests that the increase in IL-1β triggered by Aβ is mediated by JNK activation since the data presented in Fig. 3c show that treatment of cells with the JNK inhibitor D-JNKiI significantly attenuated the Aβ-induced change (+++p <  0.001; anova).

image

Figure 3.  JNK activation is required for Aβ-mediated IL-1β release. JNK phosphorylation was significantly increased in hippocampal tissue prepared from Aβ-treated rats compared with control rats; (*p < 0.05 pJNK1, **p < 0.01 pJNK2; anova, n = 5; a and b, respectively). Co-treatment of rats with IL-4 inhibited the Aβ-induced increase in pJNK2 (++p < 0.01; anova) and in pJNK1 (+p < 0.05; anova). The data are mean values obtained from densitometric analysis and are expressed as a ratio of pJNK: total JNK. Total JNK was not affected by treatment. Representative photographs are presented for phosphorylated JNK and total JNK (tJNK) in control (lane 1), Aβ (lane 2), IL-4 (lane 3) and Aβ+IL-4 (lane 4) treatment groups. (c) Cultured cortical glia were incubated in the absence or presence of Aβ (2 μmol/L) with or without a 4 h pretreatment with D-JNKiI (2 μmol/L). Supernatant was removed and used for analysis of IL-1β concentration. Aβ-induced an increase in IL-1β (***p < 0.001; anova, n = 10) which was suppressed by pre-treating cells with D-JNKi I (+++p < 0.001; anova).

Download figure to PowerPoint

Consistent with previous evidence which indicated an inverse correlation between hippocampal IL-1β concentration and LTP, we report that intracerebroventricular injection of Aβ markedly attenuated LTP and that the Aβ-induced change was antagonized by IL-4 treatment (Fig. 4a). The mean percentage changes in population epsp slope in the 2 min immediately following tetanic stimulation (compared with the mean value in the 5 min immediately prior to stimulation) were 153.4 (± 2.5; SEM) and 122.8 (± 2.1) in control- and Aβ-treated rats (***p < 0.001 ; anova) and the mean values in the rats which received IL-4 alone and in combination with Aβ were 159.9 (± 0.9) and 135.2 (± 0.8), indicating a statistically significant difference between rats which received Aβ and those which received both Aβ and IL-4 (+++p < 0.001; anova; Fig. 4b). The corresponding mean percentage changes in the last 5 min of the experiment in the corresponding treatment groups were 144.5 (± 0.7), 96.8 (± 0.8), 145.9 (± 1.3), and 141.0 (0.6); statistical analysis revealed a significant Aβ-induced inhibition (***p < 0.001; anova) and a significant difference in the values obtained from rats which were treated with Aβ and those treated with Aβ and IL-4 in combination (+++p < 0.001; anova; Fig. 4c).

image

Figure 4.  Aβ-induced inhibition of LTP is abrogated by IL-4. (a) LTP was recorded in perforant path-granule cell synapses in four groups of animals injected intracerebroventricularly with sterile water as a control (▪) (n = 6), Aβ (bsl00066) (n = 6), IL-4 (bsl00072) (n = 5) or both Aβ and IL-4 together (◆) (n = 5). (b, c) Mean percentage epsp slopes 0–2 minutes and 35–40 min following tetanic stimulation. Mean values were significantly decreased in Aβ-treated animals compared with controls (***p < 0.001; anova) 35–45 min post-HFS. This was significantly reversed by co-treatment with IL-4 (+++p < 0.001; anova).

Download figure to PowerPoint

These data suggested that the primary effect of Aβ in mediating the inhibition of LTP might be activation of microglia; if this is the case, then the effect of Aβ should be abrogated by inhibiting microglial activation with minocycline. We first analyzed the effect of minocycline treatment on hippocampal MHCII mRNA expression and IL-1β concentration to confirm its ability to attenuate microglial activation in these experimental conditions. These data indicate that the Aβ-induced statistically significant increases in MHCII mRNA expression (*p < 0.05; anova) and IL-1β concentration (**p < 0.01; anova) in hippocampal tissue which were attenuated in tissue prepared from rats that received minocycline so that the values obtained in this tissue were significantly decreased compared with that obtained from rats which received only Aβ (++p < 0.01; ++p < 0.01; anova; Figs 5a and b). These data were supported by evidence from in vitro studies which also indicated that the Aβ-induced increases in both measures were abrogated by co-treatment of cells with minocycline (Figs 5c and d).

image

Figure 5.  Microglial activation is inhibited by minocycline in vivo and in vitro. Hippocampal tissue was analyzed for IL-1β concentration by ELISA and MHCII mRNA expression, using OX-6 RT-PCR. Aβ treatment significantly increased both (a) MHCII mRNA expression (*p < 0.05; anova, n = 5) and (b) IL-1β concentration (**p < 0.01; anova, n = 7). Pretreatment of rats with minocycline (100 mg/kg/day) for 7 days reversed this Aβ-induced increase in both MHCII mRNA expression (++p < 0.01; anova) and IL-1β (++p < 0.01; anova). Cultured cortical glia were incubated in the absence or presence of Aβ (2 μmol/L) with or without minocycline pre-treatment for 1 h (30 μg/mL). Cells were analyzed for expression of MHCII mRNA using OX-6 RT-PCR (c) and supernatant was removed and used for analysis of IL-1β concentration (d). (c) Aβ increased MHCII mRNA expression and minocycline abrogated the Aβ-induced change. A representative photograph is presented to show expression of MHCII mRNA and actin for control (lane 1), Aβ (lane 2), minocycline (lane 3) and Aβ + minocycline (lane 4) treatment groups. (d) Aβ induced an increase in IL-1β (***p < 0.001; anova, n = 4) when compared with controls, which was suppressed by treatment with minocycline (+++p < 0.001; anova).

Download figure to PowerPoint

As shown in Fig. 4 (and presented here in Fig. 6 again for comparison), intracerebroventricular injection of Aβ attenuated LTP in perforant path-granule cell synapses. However, the data presented in Fig. 6 show that the effect of Aβ was attenuated by pretreatment of rats with minocycline; the mean percentage changes in population epsp slope in the 2 min immediately following tetanic stimulation (compared with the mean value in the 5 min immediately prior to stimulation) were 153.4 (± 2.5; SEM) and 122.8 (± 2.1) in control- and Aβ-treated rats (i.e., data from Fig. 4) and the mean values in the rats which received minocycline alone and in combination with Aβ were 144.5 (± 3.2) and 143.6 (± 0.83), indicating a statistically significant difference between rats which received Aβ and those which received both Aβ and minocycline (+++p < 0.001; anova; Fig. 6b). The corresponding mean percentage changes in the last 5 min of the experiment were 144.5 (± 0.7), 96.8 (± 0.8; the same as in Fig. 4), 135.8 (± 1.8) and 137.6 (0.51); statistical analysis revealed a significant Aβ-induced inhibition (***p < 0.001; anova) and a significant difference in the values obtained from rats which were treated with Aβ and those treated with Aβ and minocycline in combination (+++p < 0.001; anova; Fig. 6c).

image

Figure 6.  Aβ-induced inhibition of LTP is abrogated by minocycline. (a) Pre-treatment of rats with minocycline (100 mg/kg/day) for 7 days abrogated the deficit in LTP induced by Aβ (2 μmol/L) so that population epsp slope in response to tetanic stimulation in the rats treated with Aβ (n = 5) was significantly reduced compared with that in rats treated with saline (n = 5) or Aβ + minocycline (n = 5). (b) and (c) Analysis of the mean percentage changes in population epsp slope the 2 min immediately following tetanic stimulation (b) and in the last 5 min of the experiment (c) compared with the mean value in the 5 min prior to tetanic stimulation revealed a significant decrease in Aβ treated rats compared with other groups (***p < 0.001; anova). This was significantly reversed by co-treatment with minocycline (+++p < 0.001; anova). Values are means ± SEM. Control (▪), Aβ (bsl00066), minocycline (bsl00072), Aβ + minocycline (◆).

Download figure to PowerPoint

We assessed IL-4 concentration in tissue prepared from control-treated and minocycline-treated rats that were injected intracerebroventricularly with Aβ or vehicle. The data indicate that hippocampal IL-4 concentration was significantly increased in tissue prepared from Aβ-treated rats which received minocycline (**p < 0.01; anova; Fig. 7) and although the value was somewhat increased in tissue prepared from minocycline-treated rats, this was not statistically greater than that in tissue prepared from control-treated rats.

image

Figure 7.  Minocycline increases IL-4 in hippocampus following Aβ treatment. IL-4 concentration was assessed by ELISA in hippocampal homogenate tissue from rats treated with Aβ, minocycline or Aβ + minocycline. Mean IL-4 concentration was significantly increased in hippocampal tissue prepared from Aβ-treated rats pretreated with minocycline (100 mg/kg/day), compared with rats treated with Aβ (**p < 0.01; anova, n = 8).

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We set out to assess whether IL-4 might abrogate the inflammatory changes induced by Aβ and thereby inhibit its effect on LTP in dentate gyrus, and to establish whether activation of glial cells significantly contributed to the Aβ-induced changes. The most significant finding of this study is that treatment of rats with IL-4 antagonizes the inflammatory changes induced by intracerebroventricular injection of Aβ which leads to impairment in LTP in perforant path-granule cell synapses. The evidence highlights the role played by activated glial cells in these Aβ- induced changes.

Intracerebroventricular injection of Aβ triggers a rapid inflammatory change in hippocampus characterized by a significant increase in hippocampal concentration of IL-1β as previously reported (Minogue et al. 2003), accompanied by increases in hippocampal concentration of IFNγ, TNFα and, to a lesser extent, IL-6. The evidence couples these changes with an increase in hippocampal expression of MHCII mRNA, which is a marker of microglial activation (Frank et al. 2006), and with increased activation of JNK1 and JNK2 which is closely coupled with inhibition of LTP (Curran et al. 2003; Minogue et al. 2003; Barry et al. 2005; Nolan et al. 2005). The observed effects of Aβ on MHCII mRNA, IL-1β and IL-6 in vivo, were mirrored by similar changes in vitro.

We demonstrate that the effect of Aβ treatment on MHCII mRNA expression in hippocampus of Aβ-treated rats was attenuated by intracerebroventricular injection of IL-4. We believe these are the first data to indicate such an effect in vivo. The observation that IL-4 attenuated the Aβ-induced increase in cytokine concentration supports our earlier observations that IL-4 attenuates the age-related, IL-1β-induced and LPS-induced increases in hippocampal IL-1β concentration (Barry et al. 2005; Nolan et al. 2005). In parallel with these data, we observed that IL-4 attenuated the effect of Aβ on MHCII mRNA expression in cultured glia and therefore we suggest that IL-4 contributes to the maintenance of glial cells in a quiescent state in vivo as well as in vitro. Previous analysis of the effect of IL-4 on Aβ-induced changes in primary murine microglia revealed that IL-10, but not IL-4, inhibited the increase in IL-1β, although in the human THP-1 monocyte cell line, both anti-inflammatory cytokines blocked the Aβ-induced increases in IL-6 and TNFα, and IL-4 blocked the Aβ-induced increase in IL-1β and IL-6 (Szczepanik et al. 2001). IL-4 has also been shown to modulate Aβ-induced changes in the murine microglial cell line N9 (Iribarren et al. 2005), while its neuroprotective effect against the Aβ-induced neurodegeneration, which is accompanied by microglial activation in foetal brain cell cultures, has also been documented (Chao et al. 1994).

The present findings indicate that the inhibition of LTP induced by injection of Aβ was attenuated by IL-4. The neurotoxicity of Aβ peptides was initially associated with fibrillar forms of Aβ, such as those found in the neuritic plaques of Alzheimer’s Disease (Pike et al. 1991; Lorenzo and Yankner 1994) but it has subsequently been demonstrated that the active Aβ species are oligomeric and not monomeric or fibrillar (Walsh et al. 2002; Cleary et al. 2005; Lesne et al. 2006), although under certain circumstances, for example when the glutamate at position 22 is replaced with glycine (i.e., Arctic mutation), fibrillar Aβ is also capable of inhibiting LTP (Rowan et al. 2004). It is therefore important to acknowledge that, while IL-4 might antagonize the effects of this preparation of Aβ (a mixture of fibrillar and oligomeric forms of Aβ), systematic analysis of its effects on changes induced by specific forms of Aβ remains to be undertaken. While we have previously linked the inhibition of LTP with increased hippocampal IL-1β concentration (Minogue et al. 2003; Barry et al. 2005; Nolan et al. 2005), the present findings also link the deficit in LTP with increased TNFα, which supports the suggestion that TNFα plays a role in mediating Aβ-induced inhibition of LTP (Wang et al. 2005).

In addition to its effect on IL-1β, Aβ treatment also increased the hippocampal concentration of IFNγ and the data show that IFNγ increases MHCII expression and IL-1β concentration in vitro. IFNγ has long been identified as amongst the most potent activators of microglia (Nguyen and Benveniste 2000; Hausler et al. 2002; Gasic-Milenkovic et al. 2003; Kim et al. 2004), and interestingly, increased expression of IFNγ in transgenic mice which overexpress APP has been observed in clusters of activated microglia, astrocytes and the associated Aβ deposits (Abbas et al. 2002). Here we find that Aβ induced an increase in IFNγin vivo, and that this is attenuated by IL-4. These data are consistent with the well-described antagonistic effects of IL-4 in vitro (O’Keefe et al. 1999; Nguyen and Benveniste 2000). We report that the Aβ-induced increase in IL-1β is attenuated in glia which were incubated in the presence of anti-IFNγ antibody suggesting that IFNγ plays a role in mediating Aβ-induced changes in vitro. This finding is consistent with the report that IFNγ is released from microglia (Suzuki et al. 2005) although its release from endothelial cells in response to Aβ has been demonstrated (Suo et al. 1998).

The present data show that minocycline inhibited the Aβ-induced increase in MHCII expression in vivo and in vitro which is consistent with the reports (a) that it inhibited Aβ-induced glial activation in the rat hippocampus in vivo (Ryu et al. 2004), and (b) that it reduced the Aβ-induced release of inflammatory cytokines from human microglia (Familian et al. 2006). In addition to its effect on MHCII mRNA, we found that treatment of rats with minocycline for 7 days abrogated the Aβ-induced increase IL-1β concentration and inhibition of LTP and therefore directly couple the restoration of LTP with down-regulation of microglial activation. Minocycline treatment has also been shown to attenuate the age-related deficit in LTP and the associated increase microglial activation and IL-1β concentration (Griffin et al. 2006), while application of minocycline to hippocampal slices also abrogated the Aβ-induced inhibition of LTP (Wang et al. 2004b), indicating an acute effect of minocycline on LTP in vitro; although not explicitly shown, it was assumed that the effect was due to its ability to decrease microglial activation.

It has been assumed that the anti-inflammatory action of minocycline is a consequence of its ability to inhibit microglial activation but the involvement of T cells has been highlighted by the finding that it attenuates the increase in TNFα triggered by interaction of activated T cells with microglia (Giuliani et al. 2005). In addition, while minocycline has been shown to reduce the injury volume following middle cerebral artery occlusion in the immature brain, this was found to be independent of an effect on microglial activation (Fox et al. 2005). The present data indicate that minocycline significantly increases IL-4 concentration in hippocampus of rats treated with Aβ. Given that we show IL-4 down-regulates Aβ-induced glial activation, the possibility is raised that the ability of minocycline to ameliorate the negative impact of Aβ on LTP may be a consequence of the minocycline-driven increase in IL-4.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Funded by Science Foundation Ireland, The Health Research Board and The Higher Education Authority Ireland (PRTLI). RMC is a Trinity College Foundation Fellow.

References

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • Abbas N., Bednar I., Mix E., Marie S., Paterson D., Ljungberg A., Morris C., Winblad B., Nordberg A. and Zhu J. (2002) Up-regulation of the inflammatory cytokines IFN-gamma and IL-12 and down-regulation of IL-4 in cerebral cortex regions of APP(SWE) transgenic mice. J. Neuroimmunol. 126, 5057.
  • Barry C. E., Nolan Y., Clarke R. M., Lynch A. and Lynch M. A. (2005) Activation of c-Jun-N-terminal kinase is critical in mediating lipopolysaccharide-induced changes in the rat hippocampus. J. Neurochem. 93, 221231.
  • Blum-Degen D., Muller T., Kuhn W., Gerlach M., Przuntek H. and Riederer P. (1995) Interleukin-1 beta and interleukin-6 are elevated in the cerebrospinal fluid of Alzheimer’s and de novo Parkinson’s disease patients. Neurosci. Lett. 202, 1720.
  • Chao C. C., Hu S., Kravitz F. H., Tsang M., Anderson W. R. and Peterson P. K. (1994) Transforming growth factor-beta protects human neurons against beta-amyloid-induced injury. Mol. Chem. Neuropathol. 23, 159178.
  • Chen Q. S., Wei W. Z., Shimahara T. and Xie C. W. (2002) Alzheimer amyloid beta-peptide inhibits the late phase of long-term potentiation through calcineurin-dependent mechanisms in the hippocampal dentate gyrus. Neurobiol. Learn. Mem. 77, 354371.
  • Cleary J. P., Walsh D. M., Hofmeister J. J., Shankar G. M., Kuskowski M. A., Selkoe D. J. and Ashe K. H. (2005) Natural oligomers of the amyloid-beta protein specifically disrupt cognitive function. Nat. Neurosci. 8, 7984.
  • Curran B. P., Murray H. J. and O’Connor J. J. (2003) A role for c-Jun N-terminal kinase in the inhibition of long-term potentiation by interleukin-1beta and long-term depression in the rat dentate gyrus in vitro. Neuroscience 118, 347357.
  • Familian A., Boshuizen R. S., Eikelenboom P. and Veerhuis R. (2006) Inhibitory effect of minocycline on amyloid beta fibril formation and human microglial activation. Glia 53, 233240.
  • Fan L. W., Pang Y., Lin S., Rhodes P. G. and Cai Z. (2005) Minocycline attenuates lipopolysaccharide-induced white matter injury in the neonatal rat brain. Neuroscience 133, 159168.
  • Fox C., Dingman A., Derugin N., Wendland M. F., Manabat C., Ji S., Ferriero D. M. and Vexler Z. S. (2005) Minocycline confers early but transient protection in the immature brain following focal cerebral ischemia-reperfusion. J. Cereb. Blood Flow. Metab. 25, 11381149.
  • Frank M. G., Barrientos R. M., Biedenkapp J. C., Rudy J. W., Watkins L. R. and Maier S. F. (2006) mRNA up-regulation of MHC II and pivotal pro-inflammatory genes in normal brain aging. Neurobiol. Aging 27, 717722.
  • Freir D. B., Costello D. A. and Herron C. E. (2003) A beta 25-35-induced depression of long-term potentiation in area CA1 in vivo and in vitro is attenuated by verapamil. J. Neurophysiol. 89, 30613069.
  • Gasic-Milenkovic J., Dukic-Stefanovic S., Deuther-Conrad W., Gartner U. and Munch G. (2003) Beta-amyloid peptide potentiates inflammatory responses induced by lipopolysaccharide, interferon -gamma and ‘advanced glycation endproducts’ in a murine microglia cell line. Eur. J. Neurosci. 17, 813821.
  • Giuliani F., Hader W. and Yong V. W. (2005) Minocycline attenuates T cell and microglia activity to impair cytokine production in T cell-microglia interaction. J. Leukoc. Biol. 78, 135143.
  • Griffin W. S., Stanley L. C., Ling C., White L., MacLeod V., Perrot L. J., White C. L. III, and Araoz C. (1989) Brain interleukin 1 and S-100 immunoreactivity are elevated in Down syndrome and Alzheimer disease. Proc. Natl Acad. Sci. USA 86, 76117615.
  • Griffin R., Nally R., Nolan Y., McCartney Y., Linden J. and Lynch M. A. (2006) The age-related attenuation in long-term potentiation is associated with microglial activation. J. Neurochem. ??, ????.
  • Hausler K. G., Prinz M., Nolte C., Weber J. R., Schumann R. R., Kettenmann H. and Hanisch U. K. (2002) Interferon-gamma differentially modulates the release of cytokines and chemokines in lipopolysaccharide- and pneumococcal cell wall-stimulated mouse microglia and macrophages. Eur. J. Neurosci. 16, 21132122.
  • Iribarren P., Zhou Y., Hu J., Le Y. and Wang J. M. (2005) Role of formyl peptide receptor-like 1 (FPRL1/FPR2) in mononuclear phagocyte responses in Alzheimer disease. Immunol. Res. 31, 165176.
  • Kim H. S., Whang S. Y., Woo M. S., Park J. S., Kim W. K. and Han I. O. (2004) Sodium butyrate suppresses interferon-gamma-, but not lipopolysaccharide-mediated induction of nitric oxide and tumor necrosis factor-alpha in microglia. J. Neuroimmunol. 151, 8593.
  • Klyubin I., Walsh D. M., Cullen W. K., Fadeeva J. V., Anwyl R., Selkoe D. J. and Rowan M. J. (2004) Soluble Arctic amyloid beta protein inhibits hippocampal long-term potentiation in vivo. Eur. J. Neurosci. 19, 28392846.
  • Ledeboer A., Sloane E. M., Milligan E. D., Frank M. G., Mahony J. H., Maier S. F. and Watkins L. R. (2005) Minocycline attenuates mechanical allodynia and proinflammatory cytokine expression in rat models of pain facilitation. Pain 115, 7183.
  • Lesne S., Koh M. T., Kotilinek L., Kayed R., Glabe C. G., Yang A., Gallagher M. and Ashe K. H. (2006) A specific amyloid-beta protein assembly in the brain impairs memory. Nature 440, 352357.
  • Lorenzo A. and Yankner B. A. (1994) Beta-amyloid neurotoxicity requires fibril formation and is inhibited by congo red. Proc. Natl Acad. Sci. USA 91, 12 24312 247.
  • Lynch M. A., Errington M. L. and Bliss T. V. (1985) Long-term potentiation of synaptic transmission in the dentate gyrus: increased release of [14C]glutamate without increase in receptor binding. Neurosci. Lett. 62, 123129.
  • Lynch A. M., Moore M., Craig S., Lonergan P. E., Martin D. S. and Lynch M. A. (2003) Analysis of interleukin-1 beta-induced cell signaling activation in rat hippocampus following exposure to gamma irradiation. Protective effect of eicosapentaenoic acid. J. Biol. Chem. 278, 51 07551 084.
  • Lynch A. M., Loane D. J., Minogue A. M. and Lynch M. A. (2005) Cytokine modulation by eicosapentaenoic acid in the aged hippocampus. Abstract No. 910.4. Society for Neuroscience 2005. Washington, D.C..
  • Maher F. O., Nolan Y. and Lynch M. A. (2005) Downregulation of IL-4-induced signalling in hippocampus contributes to deficits in LTP in the aged rat. Neurobiol. Aging 26, 717728.
  • Martin D. S., Lonergan P. E., Boland B., Fogarty M. P., Brady M., Horrobin D. F., Campbell V. A. and Lynch M. A. (2002) Apoptotic changes in the aged brain are triggered by interleukin-1beta-induced activation of p38 and reversed by treatment with eicosapentaenoic acid. J. Biol. Chem. 277, 34 23934 246.
  • McGeer P. L., Schulzer M. and McGeer E. G. (1996) Arthritis and anti-inflammatory agents as possible protective factors for Alzheimer’s disease: a review of 17 epidemiologic studies. Neurology 47, 425432.
  • Minogue A. M., Schmid A. W., Fogarty M. P., Moore A. C., Campbell V. A., Herron C. E. and Lynch M. A. (2003) Activation of the c-Jun N-terminal kinase signaling cascade mediates the effect of amyloid-beta on long term potentiation and cell death in hippocampus: a role for interleukin-1beta? J. Biol. Chem. 278, 27 97127 980.
  • Nguyen V. T. and Benveniste E. N. (2000) IL-4-activated STAT-6 inhibits IFN-gamma-induced CD40 gene expression in macrophages/microglia. J. Immunol. 165, 62356243.
  • Nolan Y., Maher F. O., Martin D. S., Clarke R. M., Brady M. T., Bolton A. E., Mills K. H. and Lynch M. A. (2005) Role of interleukin-4 in regulation of age-related inflammatory changes in the hippocampus. J. Biol. Chem. 280, 93549362.
  • O’Keefe G. M., Nguyen V. T. and Benveniste E. N. (1999) Class II transactivator and class II MHC gene expression in microglia: modulation by the cytokines TGF-beta, IL-4, IL-13 and IL-10. Eur. J. Immunol. 29, 12751285.
  • Pike C. J., Walencewicz A. J., Glabe C. G. and Cotman C. W. (1991) Aggregation-related toxicity of synthetic beta-amyloid protein in hippocampal cultures. Eur. J. Pharmacol. 207, 367368.
  • Rowan M. J., Klyubin I., Wang Q. and Anwyl R. (2004) Mechanisms of the inhibitory effects of amyloid beta-protein on synaptic plasticity. Exp. Gerontol. 39, 16611667.
  • Ryu J. K., Franciosi S., Sattayaprasert P., Kim S. U. and McLarnon J. G. (2004) Minocycline inhibits neuronal death and glial activation induced by beta-amyloid peptide in rat hippocampus. Glia 48, 8590.
  • Saleshando G. and O’Connor J. J. (2000) SB203580, the p38 mitogen-activated protein kinase inhibitor blocks the inhibitory effect of beta-amyloid on long-term potentiation in the rat hippocampus. Neurosci. Lett. 288, 119122.
  • Seabrook T. J., Jiang L., Maier M. and Lemere C. A. (2006) Minocycline affects microglia activation, Abeta deposition, and behavior in APP-tg mice. Glia 53, 776782.
  • Sheng J. G., Mrak R. E., Bales K. R., Cordell B., Paul S. M., Jones R. A., Woodward S., Zhou X. Q., McGinness J. M. and Griffin W. S. (2000) Overexpression of the neuritotrophic cytokine S100beta precedes the appearance of neuritic beta-amyloid plaques in APPV717F mice. J. Neurochem. 74, 295301.
  • Suo Z., Tan J., Placzek A., Crawford F., Fang C. and Mullan M. (1998) Alzheimer’s beta-amyloid peptides induce inflammatory cascade in human vascular cells: the roles of cytokines and CD40. Brain Res. 807, 110117.
  • Suzuki Y., Claflin J., Wang X., Lengi A. and Kikuchi T. (2005) Microglia and macrophages as innate producers of interferon-gamma in the brain following infection with Toxoplasma gondii. Int. J. Parasitol. 35, 8390.
  • Szczepanik A. M., Funes S., Petko W. and Ringheim G. E. (2001) IL-4, IL-10 and IL-13 modulate A beta(1–42)-induced cytokine and chemokine production in primary murine microglia and a human monocyte cell line. J. Neuroimmunol. 113, 4962.
  • Walsh D. M., Klyubin I., Fadeeva J. V., Cullen W. K., Anwyl R., Wolfe M. S., Rowan M. J. and Selkoe D. J. (2002) Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature 416, 535539.
  • Wang H. W., Pasternak J. F., Kuo H. et al. (2002) Soluble oligomers of beta amyloid (1-42) inhibit long-term potentiation but not long-term depression in rat dentate gyrus. Brain Res. 924, 133140.
  • Wang Q., Rowan M. J. and Anwyl R. (2004a) Beta-amyloid-mediated inhibition of NMDA receptor-dependent long-term potentiation induction involves activation of microglia and stimulation of inducible nitric oxide synthase and superoxide. J. Neurosci. 24, 60496056.
  • Wang Q., Walsh D. M., Rowan M. J., Selkoe D. J. and Anwyl R. (2004b) Block of long-term potentiation by naturally secreted and synthetic amyloid beta-peptide in hippocampal slices is mediated via activation of the kinases c-Jun N-terminal kinase, cyclin-dependent kinase 5, and p38 mitogen-activated protein kinase as well as metabotropic glutamate receptor type 5. J. Neurosci. 24, 33703378.
  • Wang Q., Wu J., Rowan M. J. and Anwyl R. (2005) Beta-amyloid inhibition of long-term potentiation is mediated via tumor necrosis factor. Eur. J. Neurosci. 22, 28272832.
  • Yrjanheikki J., Keinanen R., Pellikka M., Hokfelt T. and Koistinaho J. (1998) Tetracyclines inhibit microglial activation and are neuroprotective in global brain ischemia. Proc. Natl Acad. Sci. USA 95, 15 76915 774.