Infiltrating macrophages contribute to age-related neuroinflammation in C57/BL6 mice.

The recognized role of neuroinflammation in the age-related deterioration of neuronal function highlights the importance of understanding the factors that control microglial activation. Microglia, as the immune cells of the brain, are the arbiters of the inflammatory profile in the brain. Normally they are maintained in a quiescent state by means of ligand-receptor interactions with neurons, within a prevailing anti-inflammatory microenvironment. The evidence indicates that, as the ageing process continues, microglia become activated, shift towards an inflammatory phenotype and alter the milieu in the brain. Although there has been progress in identifying factors that contribute to age-related microglial activation, our understanding remains incomplete. Here we report that there was an age-related increase in circulating inflammatory cytokines, accompanied by microglial activation. Neutrophils, and to a greater extent, macrophages, infiltrate the brain with age, perhaps as a result of increased chemokine expression in the brain, specifically CXCL1 and CCL2. We sought to determine whether macrophages might trigger microglial activation and the evidence shows that conditioned medium obtained from interferon-γ (IFNγ)-stimulated macrophages potently activated microglia. The data suggest that infiltrating macrophages may be one factor that contributes to age-related microglial activation.

Inflammatory microglia are glycolytic and iron retentive and typify the microglia in APP/PS1 mice.

Microglia, like macrophages, can adopt inflammatory and anti-inflammatory phenotypes depending on the stimulus. In macrophages, the evidence indicates that these phenotypes have different metabolic profiles with lipopolysaccharide (LPS)- or interferon-γ (IFNγ)-stimulated inflammatory cells switching to glycolysis as their main source of ATP and interleukin-4 (IL-4)-stimulated cells utilizing oxidative phosphorylation. There is a paucity of information regarding the metabolic signatures of inflammatory and anti-inflammatory microglia. Here, we polarized primary microglia with IFNγ and show that the characteristic increases in tumor necrosis factor-α (TNFα) and nitric oxide synthase 2 (NOS2) were accompanied by increased glycolysis and an increase in the expression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB)3, an enzyme that plays a significant role in driving glycolysis. These changes were associated with increased expression of ferritin and retention of iron in microglia. Significantly, retention of iron in microglia increased TNFα expression and also increased glycolysis suggesting that increased intracellular iron concentration may drive the metabolic and/or inflammatory changes. Analysis of microglia prepared from wildtype mice and from transgenic mice that overexpress amyloid precursor protein (APP) and presenilin 1 (PS1; APP/PS1) revealed genotype-related increases in glycolysis, accompanied by increased PFKFB3, and an increase in the expression of ferritin. The data indicate a distinct metabolic signature of inflammatory microglia from APP/PS1 mice that are also distinguishable by their iron handling profiles.

Inhibiting the NLRP3 inflammasome with MCC950 promotes non-phlogistic clearance of amyloid-ß and cognitive function in APP/PS1 mice.

Activation of the inflammasome is implicated in the pathogenesis of an increasing number of inflammatory diseases, including Alzheimer's disease (AD). Research reporting inflammatory changes in post mortem brain tissue of individuals with AD and GWAS data have convincingly demonstrated that neuroinflammation is likely to be a key driver of the disease. This, together with the evidence that genetic variants in the NLRP3 gene impact on the risk of developing late-onset AD, indicates that targetting inflammation offers a therapeutic opportunity. Here, we examined the effect of the small molecule inhibitor of the NLRP3 inflammasome, MCC950, on microglia in vitro and in vivo. The findings indicate that MCC950 inhibited LPS+Aß-induced caspase 1 activation in microglia and this was accompanied by IL-1ß release, without inducing pyroptosis. We demonstrate that MCC950 also inhibited inflammasome activation and microglial activation in the APP/PS1 mouse model of AD. Furthermore, MCC950 stimulated Aß phagocytosis in vitro, and it reduced Aß accumulation in APP/PS1 mice, which was associated with improved cognitive function. These data suggest that activation of the inflammasome contributes to amyloid accumulation and to the deterioration of neuronal function in APP/PS1 mice and demonstrate that blocking assembly of the inflammasome may prove to be a valuable strategy for attenuating changes that negatively impact on neuronal function.

The neural cell adhesion molecule-derived peptide, FGL, attenuates lipopolysaccharide-induced changes in glia in a CD200-dependent manner.

Fibroblast growth loop (FGL) is a neural cell adhesion molecule (NCAM)-mimetic peptide that mimics the interaction of NCAM with fibroblast growth factor receptor (FGFR). FGL increases neurite outgrowth and promotes neuronal survival in vitro, and it has also been shown to have neuroprotective effects in vivo. More recent evidence has indicated that FGL has anti-inflammatory effects, decreasing age-related changes in microglial activation and production of inflammatory cytokines. These changes have been associated with an FGL-induced increase in expression of the glycoprotein, CD200, which interacts with its receptor to help maintain microglia in a quiescent state. However whether the FGL-induced anti-inflammatory effects are CD200-dependent has not been examined. The objective of this study was to address this question. Mixed glia were prepared from brain tissue of neonatal wildtype and CD200-deficient mice and preincubated with FGL prior to stimulation with lipopolysaccharide (LPS). Cells were assessed for mRNA expression of markers of microglial activation, CD11b, CD40 and intercellular adhesion molecule 1 (ICAM-1) and also the inflammatory cytokines, interleukin (IL)-1ß, IL-6 and tumour necrosis factor (TNF)-α, while supernatant concentrations of these cytokine were also assessed. LPS significantly increased all these parameters and the effect was greater in cells prepared from CD200-deficient mice. Whereas FGL attenuated the LPS-induced changes in cells from wildtype mice, it did not do so in cells from CD200-deficient mice. We conclude that the FGL-induced changes in microglial activation are CD200-dependent and demonstrate that the interaction of astrocytes with microglia is critically important for modulating microglial activation.

Impaired performance of female APP/PS1 mice in the Morris water maze is coupled with increased Aß accumulation and microglial activation.

BACKGROUND: Alzheimer's disease (AD) is characterized by progressive neuronal loss and cognitive decline. Epidemiological studies suggest that the risk of AD is higher in women even when data are adjusted for age. OBJECTIVE: We set out to compare changes in 9-month-old male and female mice which overexpress amyloid precursor protein (APP) with presenilin (PS1; APP/PS1 mice) and to evaluate whether any changes were coupled with deficits in spatial learning. METHODS: APP/PS1 mice were assessed for their ability to learn in the Morris water maze and Aß burden assessed by Congo Red and Aß triple ultrasensitive assay. Neuroinflammatory changes were examined in brain tissue along with expression of Aß-generating and Aß-degrading enzymes. RESULTS: A deficit in reversal phase learning in the Morris water maze was observed in female mice and was paralleled by evidence of increased accumulation of Aß, microglial activation and expression of IL-1ß. Accumulation of Aß was coupled with an increase in expression of BACE-1 and a decrease in insulin-degrading enzyme (IDE). CONCLUSION: The results indicate that the observed impairment in spatial memory in female APP/PS1 mice correlated with increased Aß burden and the changes in Aß may have occurred as a result of enhanced BACE-1 and decreased IDE expression.

Bland dyskaryosis: a new pitfall in liquid-based cytology.

OBJECTIVE: To describe our experience in recognizing an unusual presentation of severe dyskaryosis at two large cytology centres using ThinPrep liquid-based cytology (LBC). LBC has been introduced in England following successful pilot studies. It is clear that LBC improves visualization and preservation of cells, and that sensitivity for high-grade dyskaryosis is at least as good as for conventional cytology, and may be better. Several variants of high-grade dyskaryosis have been described on conventional cytology, including small and pale cell dyskaryosis. These are also seen on LBC. We are reporting a new variant of dyskaryosis which in our experience is seen only in LBC specimens prepared by the Thinprep method, which we have named bland dyskaryosis. This has not to our knowledge been previously described. Bland dyskaryosis is characterized by cells with a high nuclear/cytoplasmic ratio, nuclear hyperchromasia and is present in groups with a chaotic architecture. The chromatin pattern appears bland at low power screening examination. On high power examination, however, the chromatin pattern can be seen to be subtly abnormal. Nuclear membranes are smooth. These changes mimic endocervical cells or immature squamous metaplasia at low power. METHOD: Identification and description of cytological appearances observed in routine practice and correlation with histological diagnosis. CONCLUSION: The features of bland dyskaryosis should be disseminated through teaching activities. Recognition of this previously undescribed variant will prevent false negative reporting of LBC samples.

Docosahexaenoic acid-induced changes in phospholipids in cortex of young and aged rats: a lipidomic analysis.

The age-related decline in cognitive function has been associated with biochemical changes that can be attenuated following n-3 polyunsaturated fatty acid treatment. Dietary supplementation with docosahexaenoic acid (DHA) has been shown to reverse age-related changes in synaptic function. Here, lipidomic analyses were undertaken to examine changes in lipid classes and phospholipid species in cortical tissue of young (2-4 months) and aged (20-22 months), control- and DHA-treated (10mg daily) rats following treatment for 8 weeks, aiming to explore the mechanism of DHA action. Dietary supplementation normalised the age-related decrease in unsaturation index, reduced the levels of arachidonic acid-containing phospholipids in both young and aged animals, and gave rise to production of new phosphatidylserine and phosphatidylinositol species. These findings suggest that DHA may mediate some of its effects through alterations in the membrane lipid composition that can consequently affect the production of pro-inflammatory mediators and signalling molecular species.

Evidence that vitamin D3 reverses age-related inflammatory changes in the rat hippocampus.

One of the major challenges in neuroscience is to identify the changes which accompany aging and which contribute to the well-documented age-related deterioration in cognitive function. This is a particular challenge in the light of the vast array of reported changes, which include morphological changes like synaptic and perhaps cell loss, alteration in membrane composition and the resultant changes in function of membrane proteins, modulation of the hypothalamo-pituitary axis, impaired calcium homoeostatic mechanisms, alteration in enzyme function and decreased neurotransmitter release. In the past few years, evidence suggesting that an aged brain exhibits signs of oxidative stress and inflammatory stress has been accumulating, and recent evidence using microarray analysis has added support to this view. In this paper, we provide evidence to suggest that vitamin D3 acts as an anti-inflammatory agent and reverses the age-related increase in microglial activation and the accompanying increase in IL-1beta (interleukin-1beta) concentration.

Evidence of a protective effect of phosphatidylserine-containing liposomes on lipopolysaccharide-induced impairment of long-term potentiation in the rat hippocampus.

Lipopolysaccharide (LPS) has a negative impact on long-term potentiation (LTP) in the rat hippocampus, which has been correlated with increased concentration of interleukin-1 beta (IL-1 beta) and activation of p38 and c-Jun N-terminal kinase (JNK). It has been documented that phosphatidylserine (PS)-containing liposomes induce anti-inflammatory signals and we report that pre-treatment of rats with PS liposomes prevented these LPS-induced effects while also inhibiting microglial activation. We also observed increased concentration of the anti-inflammatory cytokine interleukin-10 (IL-10), whose intracerebroventricular injection administration mimicked the effects of PS liposomes on LTP. This suggests that administration of PS liposomes protects against the deleterious effects of LPS possibly through generation of the anti-inflammatory cytokine IL-10.

Increased IL-1beta in cortex of aged rats is accompanied by downregulation of ERK and PI-3 kinase.

Ageing is accompanied by a myriad of changes, which lead to deficits in synaptic function and recent studies have identified an increase in concentration of the proinflammatory cytokine, interleukin-1beta (IL-1beta), as a factor which significantly contributes to deterioration of cell function. Here, we consider that increased IL-1beta concentration and upregulation of IL-1beta-induced cell signalling cascades may be accompanied by downregulation of survival signals, perhaps as a consequence of decreased neurotrophins-associated signalling. The data indicate that increased IL-1beta concentration was coupled with downregulation of ERK and phosphoinositide-3 kinase (PI-3 kinase) in cortical tissue prepared from aged rats. These changes could not be attributed to decreased concentration of NGF or BDNF but the evidence suggested that they may be a consequence of an age-related change in the anti-inflammatory cytokine, IL-4. Significantly, treatment of aged rats with eicosapentaenoic acid reversed the age-related increases in IL-1beta and IL-1beta-induced signalling and also the age-related changes in IL-4, ERK and PI-3 kinase.

Long-term potentiation and memory.

One of the most significant challenges in neuroscience is to identify the cellular and molecular processes that underlie learning and memory formation. The past decade has seen remarkable progress in understanding changes that accompany certain forms of acquisition and recall, particularly those forms which require activation of afferent pathways in the hippocampus. This progress can be attributed to a number of factors including well-characterized animal models, well-defined probes for analysis of cell signaling events and changes in gene transcription, and technology which has allowed gene knockout and overexpression in cells and animals. Of the several animal models used in identifying the changes which accompany plasticity in synaptic connections, long-term potentiation (LTP) has received most attention, and although it is not yet clear whether the changes that underlie maintenance of LTP also underlie memory consolidation, significant advances have been made in understanding cell signaling events that contribute to this form of synaptic plasticity. In this review, emphasis is focused on analysis of changes that occur after learning, especially spatial learning, and LTP and the value of assessing these changes in parallel is discussed. The effect of different stressors on spatial learning/memory and LTP is emphasized, and the review concludes with a brief analysis of the contribution of studies, in which transgenic animals were used, to the literature on memory/learning and LTP.

Synaptophysin immunogold labelling of synapses decreases in dentate gyrus of the hippocampus of aged rats.

Synaptophysin expression was assessed in dentate gyrus prepared from aged (22 months) and young (4 months) rats by immunoblotting and post-embedding immunolabelling at electron microscope level. Immunoblotting showed qualitatively that there was a marked reduction in synaptophysin expression in synaptosomes of aged compared with young rats. Immunogold labelling studies in the medial molecular layer of the dentate gyrus demonstrated that gold particles were restricted to pre-synaptic boutons, and were present mainly on the membranes of the synaptic vesicles or occasionally inside vesicles. In aged rats, immunolabelling patterns and the density of immunogold particles per pre-synaptic bouton were almost 50% lower than in younger rats. However, because boutons were larger in older rats the actual labelling density per unit area of bouton (3.77) was even lower than in the young rats (7.74). The role of synaptophysin in neural plasticity and ageing should be further examined.

Chronic brain inflammation results in cell loss in the entorhinal cortex and impaired LTP in perforant path-granule cell synapses.

Alzheimer's disease (AD) is characterized by chronic neuroinflammation, significant temporal lobe cell loss, and dementia. We investigated the influence of chronic neuroinflammation produced by chronic infusion of lipopolysaccharide (LPS) into the fourth ventricle for 4 weeks upon the induction and maintenance of long-term potentiation (LTP) in the dentate gyrus of the hippocampus, a well-characterized model of cellular synaptic plasticity. We also examined for pyramidal cell loss within the entorhinal cortex an area of the brain that contains the cell bodies of the perforant path. The results demonstrate that chronic neuroinflammation results in the loss of pyramidal cells within layers II and III of the entorhinal cortex and a significant attenuation of LTP within the dentate gyrus. Similar changes may underlie the temporal lobe pathology and dementia associated with AD.

Long-term potentiation in aged rats is restored when the age-related decrease in polyunsaturated fatty acid concentration is reversed.

Several age-related changes have been identified in rat hippocampus; among these are deficits in glutamate release and long-term potentiation in dentate gyrus. These deficits correlate with a decrease in the concentration of arachidonic acid in hippocampus. In this study, the effects of dietary supplementation for 8 weeks with omega -6 or omega -3 fatty acids were assessed in groups of aged and young rats. The data presented indicate that dietary supplementation in aged rats restored the concentrations of arachidonic acid and docosahexanoic acid in hippocampal preparations to those observed in tissue prepared from young rats. In parallel, aged rats which received the experimental diets sustained long-term potentiation in a manner indistinguishable from young rats. The evidence presented supports the view that an age-related increase in reactive oxygen species production is linked with the decrease in polyunsaturated fatty acids and that a diet enriched in eicosapentanoic acid has antioxidant properties which may play a key role in reversal of the observed age-related deficits.

Analysis of the presynaptic signaling mechanisms underlying the inhibition of LTP in rat dentate gyrus by the tyrosine kinase inhibitor, genistein.

A great deal of recent evidence points to a role for tyrosine kinase in expression of LTP. Data have been presented that are consistent with the idea that tyrosine phosphorylation of proteins occurs in both the presynaptic and postsynaptic areas. In this study, we set out to investigate the role that tyrosine kinase might play presynaptically to modulate release of glutamate in an effort to understand the mechanism underlying the persistent increase in release that accompanies LTP in perforant path-granule cell synapses. We report that LTP was associated with increased calcium influx and glutamate release. LTP was also associated with an increase in phosphorylation of the alpha-subunit of calcium channels and ERK in synaptosomes prepared from dentate gyrus, and these effects were inhibited when LTP was blocked by the tyrosine kinase inhibitor, genistein. LTP was accompanied by increased protein synthesis and increased phosphorylation of CREB in entorhinal cortex, effects that were also blocked by genistein. We conclude that tetanic stimulation leads to enhanced tyrosine phosphorylation of certain presynaptically located proteins that modulate glutamate release and contribute to expression of LTP.

Long-term potentiation and spatial learning are associated with increased phosphorylation of TrkB and extracellular signal-regulated kinase (ERK) in the dentate gyrus: evidence for a role for brain-derived neurotrophic factor.

In this study, the authors investigate changes in the presynaptic terminal of the dentate gyrus that accompany 2 types of hippocampal-dependent plasticity: spatial learning and long-term potentiation (LTP). Parallel changes occurred in the dentate gyrus of rats that had undergone training in the Morris water maze and had sustained LTP. In both cases, KCl-induced brain-derived neurotrophic factor release was increased, and this was accompanied by increased phosphorylation of TrkB and the mitogen-activated protein kinase, ERK. Glutamate release was also enhanced, and the data suggest that this may be a consequence of increased activation of TrkB and ERK. Because the data indicate that similar cellular modifications are shared by these 2 forms of plasticity, they provide circumstantial evidence that LTP satisfies some of the requirements of a memory-inducing cellular substrate.

The anti-inflammatory cytokine, interleukin (IL)-10, blocks the inhibitory effect of IL-1 beta on long term potentiation. A role for JNK.

Several effects of the proinflammatory cytokine, interleukin-1 beta (IL-1 beta), have been described in the central nervous system, and one area of the brain where marked changes have been reported is the hippocampus. Among these changes are an IL-1 beta-induced inhibition of long term potentiation (LTP) in perforant path-granule cell synapses and an attenuation of glutamate release in synaptosomes prepared from the hippocampus. Evidence suggests that, at least in circulating cells, the anti-inflammatory cytokine, IL-10, antagonizes certain effects of IL-1. We investigated the effect of IL-10 on IL-1 beta-induced inhibition of LTP and glutamate release. The evidence presented indicates that IL-1 beta stimulates the stress-activated protein kinase, c-Jun-activated protein kinase (JNK), and IL-1 receptor-associated kinase, which may explain its inhibitory effect on release and LTP, and that IL-10 reversed the IL-1 beta-induced stimulation of JNK activity and inhibition of release and LTP. We observed that IL-10 abrogated the stimulatory effect of IL-1 beta on superoxide dismutase activity and reactive oxygen species production, whereas the H(2)O(2)-induced inhibition of LTP was also blocked by IL-10. We present evidence that suggests that the action of IL-10 may be mediated by its ability to induce shedding of the IL-1 type I receptor.

Identification of genetic alterations in the TGFbeta type II receptor gene promoter.

Modifications in the control sequences of tumor suppressor genes have been found to play a role in the activation or inactivation of these genes and may play an important role in tumorigenesis. For example, hypermethylation of CpG islands and promoter polymorphisms have been found to be involved in transcriptional repression. A decrease in the levels of expression of one such tumor suppressor gene, the TGFbeta type II receptor (TbetaR-II), has been associated with increased tumorigenicity in a number of human tumors. Genetic alterations have been described in several tumor types in the coding region of this gene. However, no comprehensive search for genetic alterations in the TbetaR-II promoter has been reported. Genetic alterations in the promoter of the TbetaR-II gene could inhibit binding of putative regulatory factors. For example, we have reported a A-364-G alteration in the TbetaR-II promoter, which results in decreased transcriptional activity. In this study, we analyzed the 1.0kb region upstream of the TbetaR-II transcriptional start site and found genetic alterations in 46% of the head and neck squamous cell carcinoma (SqCC) samples examined. The most frequent alteration was a G-875-A alteration, present in 41.6% of the samples. Analysis of normal healthy individuals showed a similar frequency of this alteration, suggesting that alterations within the TbetaR-II promoter are unlikely to account for the decreased expression of TbetaR-II in head and neck SqCC.

Synthesis, biological activity and comparative analysis of DNA binding affinities and human DNA topoisomerase I inhibitory activities of novel 12-alkoxy-benzo[c]phenanthridinium salts.

New antitumor 12-alkoxy-benzo[c]phenanthridinium derivatives were obtained in high yields through multistep syntheses. Analysis of DNA binding and human DNA topoisomerase I inhibitory activities demonstrates that new compounds, combining 2, 6, and 12 substitutions, interact strongly with DNA and exhibit important topoisomerase I inhibition. The cytotoxicities against solid tumor cell lines are also determined and compared with those for fagaronine and ethoxidine.

Responsiveness to transforming growth factor-beta (TGF-beta)-mediated growth inhibition is a function of membrane-bound TGF-beta type II receptor in human breast cancer cells.

Transforming growth factor-beta (TGF-beta) is a potent inhibitor of growth and proliferation of breast epithelial cells, and loss of sensitivity to its effects has been associated with malignant transformation and tumorigenesis. The biological effects of TGF-beta are mediated by the TGF-beta receptor complex, a multimer composed of TGF-beta receptor type I (TbetaR-I) and TGF-beta receptor type II (TbetaR-II) subunits. Evidence suggests that loss of expression of Tbeta3R-II is implicated in the loss of sensitivity of tumorigenic breast cell lines to TGF-beta-mediated growth inhibition. A panel of human breast cell lines, including the immortalized MCF-10F and tumorigenic MCF-7, ZR75-1, BT474, T47-D, MDA-MB231, BT20, and SKBR-3 cell lines, was characterized for responsiveness to TGF-beta-induced G1 growth arrest. Only the nontumorigenic MCF-10F and the tumorigenic MDA-MB231 cell lines demonstrated a significant inhibitory response to TGF-beta1 and a significant binding of 125I-labeled TGF-beta ligand. While expression of TbetaR-I mRNA was similar across the panel of cell lines, TbetaR-II mRNA expression was decreased significantly in all seven tumorigenic cell lines in comparison with the nontumorigenic MCF- 10F cell line. When total cellular protein was fractionated by centrifugation, TbetaR-I protein was observed in both the cytosolic and membrane fractions at similar levels in all cell lines; however, TbetaR-II protein was present in the cytosolic fraction in all cell lines, but was observed in the membrane fraction of only the TGF-beta-responsive MCF-10F and MDA-MB231 cells. Thus, lack of membrane-bound TbetaR-II protein appears to be an important determinant of resistance to TGF-beta-mediated growth inhibition in this group of breast cell lines.