Angela M. Floden

β-Amyloid-Stimulated Microglia Induce Neuron Death via Synergistic Stimulation of Tumor Necrosis Factor α and NMDA Receptors

Although abundant reactive microglia are found associated with β-amyloid (Aβ) plaques in Alzheimers disease (AD) brains, their contribution to cell loss remains speculative. A variety of studies have documented the ability of Aβ fibrils to directly stimulate microglia in vitro to assume a neurotoxic phenotype characterized by secretion of a plethora of proinflammatory molecules. Collectively, these data suggest that activated microglia play a direct role in contributing to neuron death in AD rather than simply a role in clearance after plaque deposition. Although it is clear the Aβ-stimulated microglia acutely secrete toxic oxidizing species, the identity of longer-lived neurotoxic agents remains less defined. We used Aβ-stimulated conditioned media from primary mouse microglia to identify more stable neurotoxic secretions. The NMDA receptor antagonists memantine and 2-amino-5-phosphopetanoic acid as well as soluble tumor necrosis factor α (TNFα) receptor protect neurons from microglial-conditioned media-dependent death, implicating the excitatory neurotransmitter glutamate and the proinflammatory cytokine TNFα as effectors of microglial-stimulated death. Neuron death occurs in an oxidative damage-dependent manner, requiring activity of inducible nitric oxide synthase. Toxicity results from coincident stimulation of the TNFα and NMDA receptors, because stimulations of either alone are insufficient to initiate cell death. These findings suggest the hypothesis that AD brains provide the appropriate microglial-mediated inflammatory environment for TNFα and glutamate to synergistically stimulate toxic activation of their respective signaling pathways in neurons as a contributing mechanism of cell death.

β-Amyloid Stimulates Murine Postnatal and Adult Microglia Cultures in a Unique Manner

Reactive microglia are commonly observed in association with the β-amyloid (Aβ) plaques of Alzheimers disease brains. This localization supports the hypothesis that Aβ is a specific activating stimulus for microglia. A variety of in vitro studies have used postnatal derived rodent microglia cultures to characterize the ability of Aβ to stimulate these cells. However, it is unclear whether this paradigm accurately models conditions in aged animals. To determine whether Aβ stimulatory phenotypes differ between young and adult microglia, we established cultures of acutely isolated adult murine cortical microglia to compare with postnatal derived microglial cultures. Although cells from both ages expressed robust immunoreactivity for CD68 and CD11b, their responses to activating stimuli differed. Fibrillar Aβ was rapidly phagocytosed by postnatal microglia and both oligomeric and fibrillar peptide stimulated increased tumor necrosis factor α (TNFα) secretion. However, Aβ oligomers but not fibrils stimulated TNFα secretion from adult microglia. More importantly, adult microglia had diminished ability to phagocytose Aβ fibrils. These findings demonstrate that adult microglia respond to Aβ fibril stimulation uniquely from postnatal cells and suggest that adult rather than postnatal microglia cultures are more appropriate for modeling proinflammatory changes in the aged CNS.

α-Synuclein Expression Modulates Microglial Activation Phenotype

Recent Parkinsons disease research has focused on understanding the function of the cytosolic protein, α-synuclein, and its contribution to disease mechanisms. Within neurons, α-synuclein is hypothesized to have a role in regulating synaptic plasticity, vesicle release, and trafficking. In contrast, glial-expressed α-synuclein remains poorly described. Here, we examine the consequence of a loss of α-synuclein expression on microglial activation. Using a postnatal brain-derived culture system, we defined the phenotype of microglia from wild-type and knock-out α-synuclein mice (Scna−/−). Scna−/− microglia displayed a basally increased reactive phenotype compared with the wild-type cells and an exacerbated reactive phenotype after stimulation. They also exhibited dramatic morphologic differences compared with wild-type, presenting as large, ramified cells filled with vacuole-like structures. This corresponded with increased protein levels of activation markers, CD68 and β1 integrin, in the Scna−/− cells. More importantly, Scna−/− microglia, after stimulation, secreted elevated levels of proinflammatory cytokines, TNFα (tumor necrosis factor α) and IL-6 (interleukin-6), compared with wild type. However, despite the reactive phenotype, Scna−/− cells had impaired phagocytic ability. We demonstrate for the first time that α-synuclein plays a critical role in modulating microglial activation state. We suggest that altered microglial α-synuclein expression will affect their phenotype as has already been demonstrated in neurons. This has direct ramifications for the contribution of microglia to the pathophysiology of disease, particularly in familial cases linked to altered α-synuclein expression.

Amyloid precursor protein and proinflammatory changes are regulated in brain and adipose tissue in a murine model of high fat diet-induced obesity.

Background Middle age obesity is recognized as a risk factor for Alzheimers disease (AD) although a mechanistic linkage remains unclear. Based upon the fact that obese adipose tissue and AD brains are both areas of proinflammatory change, a possible common event is chronic inflammation. Since an autosomal dominant form of AD is associated with mutations in the gene coding for the ubiquitously expressed transmembrane protein, amyloid precursor protein (APP) and recent evidence demonstrates increased APP levels in adipose tissue during obesity it is feasible that APP serves some function in both disease conditions. Methodology/Principal Findings To determine whether diet-induced obesity produced proinflammatory changes and altered APP expression in brain versus adipose tissue, 6 week old C57BL6/J mice were maintained on a control or high fat diet for 22 weeks. Protein levels and cell-specific APP expression along with markers of inflammation and immune cell activation were compared between hippocampus, abdominal subcutaneous fat and visceral pericardial fat. APP stimulation-dependent changes in macrophage and adipocyte culture phenotype were examined for comparison to the in vivo changes. Conclusions/Significance Adipose tissue and brain from high fat diet fed animals demonstrated increased TNF-α and microglial and macrophage activation. Both brains and adipose tissue also had elevated APP levels localizing to neurons and macrophage/adipocytes, respectively. APP agonist antibody stimulation of macrophage cultures increased specific cytokine secretion with no obvious effects on adipocyte culture phenotype. These data support the hypothesis that high fat diet-dependent obesity results in concomitant pro-inflammatory changes in brain and adipose tissue that is characterized, in part, by increased levels of APP that may be contributing specifically to inflammatory changes that occur.

Tumor necrosis factor alpha stimulates NMDA receptor activity in mouse cortical neurons resulting in ERK-dependent death

Multiple cytokines are secreted in the brain during pro‐inflammatory conditions and likely affect neuron survival. Previously, we demonstrated that glutamate and tumor necrosis factor alpha (TNFα) kill neurons via activation of the N‐methyl‐d‐aspartate (NMDA) and TNFα receptors, respectively. This report continues characterizing the signaling cross‐talk pathway initiated during this inflammation‐related mechanism of death. Stimulation of mouse cortical neuron cultures with TNFα results in a transient increase in NMDA receptor‐dependent calcium influx that is additive with NMDA stimulation and inhibited by pre‐treatment with the NMDA receptor antagonist, dl‐2‐amino‐5‐phosphonovaleric acid, or the α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate/kainate receptor antagonist, 6,7‐dinitroquinoxaline‐2,3‐dione. Pre‐treatment with N‐type calcium channel antagonist, ω‐conotoxin, or the voltage‐gated sodium channel antagonist, tetrodotoxin, also prevents the TNFα‐stimulated calcium influx. Combined TNFα and NMDA stimulation results in a transient increase in activity of extracellular signal‐regulated kinases (ERKs) and c‐Jun N‐terminal kinases (JNKs). Specific inhibition of ERKs but not JNKs is protective against TNFα and NMDA‐dependent death. Death is mediated via the low‐affinity TNFα receptor, TNFRII, as agonist antibodies for TNFRII but not TNFRI stimulate NMDA receptor‐dependent calcium influx and death. These data demonstrate how microglial pro‐inflammatory secretions including TNFα can acutely facilitate glutamate‐dependent neuron death.

Amyloid-β oligomers stimulate microglia through a tyrosine kinase dependent mechanism

Alzheimers disease (AD) has been well characterized by the presence of reactive microglia, often associated with β-amyloid (Aβ) plaque deposition. The oligomeric form of Aβ peptide (Aβ(o)) has neurotoxic effects in the presence of microglia and is suggested to potentiate proinflammatory changes in microglia in AD. Primary murine microglia cultures stimulated with Aβ(o) displayed increased protein phosphotyrosine and secreted tumor necrosis factor (TNF)-α levels which were attenuated by the Src/Abl inhibitor, dasatinib. Intracerebroventricular infusions of Aβ(o) into C57BL6/J mice stimulated increased microgliosis and protein phosphotyrosine levels that were also attenuated by dasatinib administration. The rodent findings were validated in human AD brains versus age-matched controls demonstrating reactive microglial association with Aβ(o) deposits and increased microglial protein phosphotyrosine and phospho-Src levels. These data suggest a role for Aβ(o) in microglial activation through a tyrosine kinase-dependant pathway both in rodent models and human disease. Use of a selective nonreceptor tyrosine kinase inhibitor such as dasatinib to attenuate microglial-dependent proinflammatory changes may prove to be an important step toward developing anti-inflammatory treatments for AD.

Microglia Demonstrate Age-Dependent Interaction with Amyloid-β Fibrils

Alzheimers disease (AD) is an age-associated disease characterized by increased accumulation of extracellular amyloid-β (Aβ) plaques within the brain. Histological examination has also revealed profound microglial activation in diseased brains often in association with these fibrillar peptide aggregates. The paradoxical presence of increased, reactive microglia yet accumulating extracellular debris suggests that these cells may be phagocytically compromised during disease. Prior work has demonstrated that primary microglia from adult mice are unable to phagocytose fibrillar Aβ1-42 in vitro when compared to microglia cultured from early postnatal animals. These data suggest that microglia undergo an age-associated decrease in microglial ability to interact with Aβ fibrils. In order to better define a temporal profile of microglia-Aβ interaction, acutely isolated, rather than cultured, microglia from 2 month, 6 month, and postnatal day 0 C57BL/6 mice were compared. Postnatal day 0 microglia demonstrated a CD47 dependent ability to phagocytose Aβ fibrils that was lost by 6 months. This corresponded with the ability of postnatal day 0 but not adult microglia to decrease Aβ immunoreactive plaque load from AD sections in vitro. In spite of limited Aβ uptake ability, adult microglia had functional phagocytic uptake of bacterial bioparticles and demonstrated the ability to adhere to both Aβ plaques and in vitro fibrillized Aβ. These data demonstrate a temporal profile of specifically Aβ-microglia interaction with a critical developmental period at 6 months in which cells remain able to interact with Aβ fibrils but lose their ability to phagocytose it.

Acetate supplementation reduces microglia activation and brain interleukin-1β levels in a rat model of Lyme neuroborreliosis

BackgroundWe have found that acetate supplementation significantly reduces neuroglia activation and pro-inflammatory cytokine release in a rat model of neuroinflammation induced with lipopolysaccharide. To test if the anti-inflammatory effect of acetate supplementation is specific to a TLR4-mediated injury, we measured markers of neuroglia activation in rats subjected to B. burgdorferi-induced neuroborreliosis that is mediated in large part by a TLR2-type mechanism.MethodsIn this study, rats were subjected to Lyme neuroborreliosis following an intravenous infusion of B. burgdorferi (B31-MI-16). Acetate supplementation was induced using glyceryl triacetate (6g/kg) by oral gavage. Immunohistochemistry, qPCR, and western blot analyses were used to measure bacterial invasion into the brain, neuroglial activation, and brain and circulating levels of interleukin 1β. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by a Tukey’s post hoc tests or using a Student’s t test assuming unequal variances when appropriate.ResultsWe found that acetate supplementation significantly reduced microglia activation by 2-fold as determined by immunohistochemical and western blot analysis. Further, acetate supplementation also reduced the expression of the pro-inflammatory cytokine IL-1β by 2-fold as compared to controls. On the other hand, the inoculation of rats with B. burgdorferi had no effect on astroglial activation as determined by immunocytochemistry and western blot analysis despite significant increases in circulation levels of antigen toward B. burgdorferi and presence of the bacteria in the central nervous system.ConclusionsThese results suggest that microglial activation is an essential component to neuroborreliosis and that acetate supplementation may be an effective treatment to reduce injury phenotype and possibly injury progression in Lyme neuroborreliosis.

BB0347, from the lyme disease spirochete Borrelia burgdorferi, is surface exposed and interacts with the CS1 heparin-binding domain of human fibronectin.

The causative agent of Lyme disease, Borrelia burgdorferi, codes for several known fibronectin-binding proteins. Fibronectin a common the target of diverse bacterial pathogens, and has been shown to be essential in allowing for the development of certain disease states. Another borrelial protein, BB0347, has sequence similarity with these other known fibronectin-binding proteins, and may be important in Lyme disease pathogenesis. Herein, we perform an initial characterization of BB0347 via the use of molecular and biochemical techniques. We found that BB0347 is expressed, produced, and presented on the outer surface of intact B. burgdorferi. We also demonstrate that BB0347 has the potential to be important in Lyme disease progression, and have begun to characterize the nature of the interaction between human fibronectin and this bacterial protein. Further work is needed to define the role of this protein in the borrelial infection process.

The multifaceted responses of primary human astrocytes and brain microvascular endothelial cells to the Lyme disease spirochete, Borrelia burgdorferi.

The vector-borne pathogen, Borrelia burgdorferi, causes a multi-system disorder including neurological complications. These neurological disorders, collectively termed neuroborreliosis, can occur in up to 15% of untreated patients. The neurological symptoms are probably a result of a glial-driven, host inflammatory response to the bacterium. However, the specific contributions of individual glial and other support cell types to the pathogenesis of neuroborreliosis are relatively unexplored. The goal of this project was to characterize specific astrocyte and endothelial cell responses to B. burgdorferi. Primary human astrocytes and primary HBMEC (human brain microvascular endothelial cells) were incubated with B. burgdorferi over a 72-h period and the transcriptional responses to the bacterium were analyzed by real-time PCR arrays. There was a robust increase in several surveyed chemokine and related genes, including IL (interleukin)-8, for both primary astrocytes and HBMEC. Array results were confirmed with individual sets of PCR primers. The production of specific chemokines by both astrocytes and HBMEC in response to B. burgdorferi, including IL-8, CXCL-1, and CXCL-10, were confirmed by ELISA. These results demonstrate that primary astrocytes and HBMEC respond to virulent B. burgdorferi by producing a number of chemokines. These data suggest that infiltrating phagocytic cells, particularly neutrophils, attracted by chemokines expressed at the BBB (blood–brain barrier) may be important contributors to the early inflammatory events associated with neuroborreliosis.