Antibody-Mediated Inhibition of Integrin α5β1 Blocks Neurotoxic Prion Peptide PrP(106-126)-Induced Activation of BV2 Microglia

Microglial activation is a characteristic feature of the pathogenesis of prion diseases. The identification of cell surface molecules that mediate the prion protein (PrP) synthetic peptide interaction with microglia is of great significance as it represents potential target molecules to modulate the events leading to the pathophysiology of prion diseases. Here, we carried out in vitro experiments to investigate the involvement of α5β1 integrin in neurotoxic prion peptide PrP(106-126)-induced activation of BV2 microglia. The results showed that the exposure to PrP(106-126) upregulated the mRNA expression of proinflammatory factors (IL-1 β, IL-6, and iNOS) and NALP3 inflammasome components (NALP3 and ASC), increased the release of iNOS and its product nitric oxide, and stimulated NF-κB activation. Blockade of α5β1 integrin with monoclonal antibody BMC5 prior to PrP(106-126) treatment abrogated the upregulation of the mRNA expression of IL-1 β, IL-6, iNOS, and ASC, but had no effect on the mRNA expression of NALP3, blocked the release of iNOS and nitric oxide, and inhibited NF-κB activation. These results suggest that α5β1 integrin is involved in the PrP(106-126)-induced microglial activation through the participation in the activation of NF-κB and NALP3/ASC inflammasome. Our study unveils a previously unidentified role of α5β1 integrin as an intermediate signaling molecule in neurotoxic prion peptides-microglia interactions and identifies a potential molecular target for the modulation of prion-induced microglial activation.     

The role of cyclo-oxygenase 1 and 2 activity in prostaglandin E(2) secretion by cultured human adult microglia: implications for Alzheimer's disease

Microglial cyclo-oxygenase (COX) expression is considered to be important in the pathogenesis of Alzheimer's disease (AD) and, therefore, constitutes a key target for therapeutic intervention. We investigated the influence of AD plaque associated factors on COX-1 and COX-2 expression and activity in adult human microglial cells in vitro. COX-2 immunoreactivity and mRNA were induced by lipopolysaccharide (LPS), not by AD plaque associated cytokines interleukin (IL)-1alpha, IL-1beta, IL-6, tumor necrosis factor (TNF)-alpha, or amyloid (A)beta(1-42). To assess functional COX activity, the release of PGE(2) into the culture medium was determined. LPS and also arachidonic acid (AA) dose-dependently stimulated PGE(2) release. The effects of AA are independent from induction of COX mRNA expression, or of de novo protein synthesis. No effects of either plaque-associated cytokines or Abeta(1-42) on PGE(2) secretion were seen, even when cells were co-stimulated with AA, to provide enough substrate. COX isotype selective inhibitors were used to discern relative contributions of COX-1 and COX-2 activities to microglial PGE(2) secretion. COX-2 and in part COX-1-selective inhibitors inhibited LPS-induced PGE(2) secretion, whereas the AA-induced PGE(2) secretion was reduced by COX-1-selective inhibitors only. Apparently, adult human microglia in vitro (1) constitutively express COX-1, and (2) do not express COX-2 upon exposure to either Abeta or plaque associated cytokines. In the light of microglial COX activity as a potential therapeutical target in AD, the data presented in this study suggest that classical NSAIDs, rather than selective COX-2 inhibitors, are more potent in reducing microglial prostaglandin secretion.     

Activation of human microglia by fibrillar prion protein-related peptides is enhanced by amyloid-associated factors SAP and C1q

Complement activation products C1q and C3d, serum amyloid P component (SAP) and activated glial cells accumulate in amyloid deposits of conformationally changed prion protein (PrP(Sc)) in Creutzfeldt-Jakob disease, Gerstmann-Str?ussler-Scheinker disease and scrapie-infected mouse brain. Biological properties, including the potential to activate microglia, relate to prion (PrP) peptide fibrillogenic abilities. We investigated if SAP and C1q influence the fibrillogenic properties of human and mouse PrP peptide and concomitantly their stimulatory effects on human microglia in vitro. PrP-peptide induced microglial IL-6 and TNF-alpha release significantly increased in the presence of SAP and C1q. Also, SAP and C1q enhanced PrP-peptide fibril formation as revealed by electron microscopy and thioflavin S-based quantitative assays. This suggests that SAP and C1q contribute to fibrillar state-dependent cellular effects of PrP. Combined, ultrastructural and thioflavin assays, together with microglial cytokine release measurements, provide a test system to screen potential, fibrillarity impeding therapeutics for prion disease.     

ERK1/2 and p38 MAP kinases control prion protein fragment 90-231-induced astrocyte proliferation and microglia activation

Astrogliosis and microglial activation are a common feature during prion diseases, causing the release of chemoattractant and proinflammatory factors as well as reactive free radicals, involved in neuronal degeneration. The recombinant protease-resistant domain of the prion protein (PrP90-231) displays in vitro neurotoxic properties when refolded in a beta-sheet-rich conformer. Here, we report that PrP90-231 induces the secretion of several cytokines, chemokines, and nitric oxide (NO) release, in both type I astrocytes and microglial cells. PrP90-231 elicited in both cell types the activation of ERK1/2 MAP kinase that displays, in astrocytes, a rapid kinetics and a proliferative response. Conversely, in microglia, PrP90-231-dependent MAP kinase activation was delayed and long lasting, inducing functional activation and growth arrest. In microglial cells, NO release, dependent on the expression of the inducible NO synthase (iNOS), and the secretion of the chemokine CCL5 were Ca(2+) dependent and under the control of the MAP kinases ERK1/2 and p38: ERK1/2 inhibition, using PD98059, reduced iNOS expression, while p38 blockade by PD169316 inhibited CCL5 release. In summary, we demonstrate that glial cells are activated by extracellular misfolded PrP90-231 resulting in a proliferative/secretive response of astrocytes and functional activation of microglia, both dependent on MAP kinase activation. In particular, in microglia, PrP90-231 activated a complex signalling cascade involved in the regulation of NO and chemokine release. These data argue in favor of a causal role for misfolded prion protein in sustaining glial activation and, possibly, glia-mediated neuronal death.     

Aspirin inhibits cytotoxicity of prion peptide PrP106-126 to neuronal cells associated with microglia activation in vitro

The synthetic peptide consisting of amino acid residues 106-126 of the human prion protein PrP106-126 has been demonstrated to generate fibrils, which damage neurons either directly by interacting with components of the cell surface to trigger cell apoptosis signaling or indirectly by activating microglia to produce inflammatory mediators. In our study, rat microglia cells were treated with PrP106-126 or scramble PrP106-126 (Scr PrP). Activated nuclear factor kappaB (NF-kappaB) was determined using immunofluorescence staining and the expression of TNF-alpha and IL-1beta mRNA was measured by quantitative RT-PCR. Inhibitory activity of aspirin on neurotoxicity of PrP106-126 associated with microglia activation was determined using an apoptosis detection kit. Treatment of microglia with 25 microM PrP106-126, but not Scr PrP, resulted in activation and translocation of NF-kappaB, which peaked after 20 min of treatment. The activation of NF-kappaB was followed by increased mRNA expression of TNF-alpha and IL-1beta peaking at about 20 h. In the presence of microglia, aspirin significantly inhibited neuro-2a cell death induced by PrP106-126. The number of neuro-2a cells in apoptosis and necrosis with 5 mM aspirin was about 3-fold lower than the cell culture without aspirin (P<0.05). These data suggest that increased production of cytokines by microglia cells in prion disease is probably regulated by NF-kappaB translocation and may contribute to neurotoxicity of prions, and neurotoxicity of PrP106-126 may be inhibited by aspirin.     

Cyclooxygenase expression in microglia and neurons in Alzheimer's disease and control brain

Epidemiological studies suggest that non-steroidal anti-inflammatory drugs (NSAIDs) lower the risk of developing Alzheimer's disease (AD). Most NSAIDs act upon local inflammatory events by inhibiting the expression or activation of cylooxygenase (COX). In the present study the expression of COX-1 and COX-2 in AD and non-demented control temporal and frontal cortex was investigated using immunohistochemistry. COX-1 expression was detected in microglial cells, while COX-2 expression was found in neuronal cells. In AD brains, COX-1-positive microglial cells were primarily associated with amyloid beta plaques, while the number of COX-2-positive neurons was increased compared to that in control brains. No COX expression was detected in astrocytes. In vitro, primary human microglial and astrocyte cultures, and human neuroblastoma cells (SK-N-SH) were found to secrete prostaglandin E2 (PGE2), especially when stimulated. PGE2 synthesis by astrocytes and SK-N-SH cells was stimulated by interleukin-1beta. Microglial cell PGE2 synthesis was stimulated by lipopolysaccharide only. Although astrocytes are used in studies in vitro to investigate the role of COX in AD, there are no indications that these cells express COX-1 or COX-2 in vivo. The different distribution patterns of COX-1 and COX-2 in AD could implicate that these enzymes are involved in different cellular processes in the pathogenesis of AD.     

Involvement of microglia-neuron interactions in the tumor necrosis factor-alpha release,microglial activation,and neurodegeneration induced by trimethyltin

Trimethyltin (TMT) is a neurotoxicant known to induce early microglial activation. The present study was undertaken to investigate the role played by these microglial cells in the TMT-induced neurotoxicity. The effects of TMT were investigated in monolayer cultures of isolated microglia or in neuron-enriched cultures and in neuron-microglia and astrocyte-microglia cocultures. The end points used were morphological criteria; evaluation of cell death and cell proliferation; and measurements of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and nitric oxide (NO) release in culture supernatant. The results showed that, in cultures of microglia, TMT (10(-6) M) caused, after a 5-day treatment, an increased release of TNF-alpha, without affecting microglial shape or cell viability. When microglia were cocultured with astrocytes, TNF-alpha release was decreased to undetectable levels. In contrast, in neuron-microglia cocultures, TNF-alpha levels were found to increase at lower concentrations of TMT (i.e., 10(-8) M). Moreover, at 10(-6) M of TMT, microglia displayed further morphological activation, as suggested by process retraction and by decrease in cell size. No morphological activation was observed in cultures of isolated microglial cells and in astrocyte-microglia cocultures. With regard to neurons, 10(-6) M of TMT induced about 30% of cell death, when applied to neuron-enriched cultures, whereas close to 100% of neuronal death was observed in neuron-microglia cocultures. In conclusion, whereas astrocytes may rather dampen the microglial activation by decreasing microglial TNF-alpha production, neuronal-microglial interactions lead to enhanced microglial activation. This microglial activation, in turn, exacerbates the neurotoxic effects of TMT. TNF-alpha may play a major role in such cell-cell communications.     

Paracetamol effectively reduces prostaglandin E2 synthesis in brain macrophages by inhibiting enzymatic activity of cyclooxygenase but not phospholipase and prostaglandin E synthase

Epidemiological studies indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) are neuroprotective, although the mechanisms underlying their beneficial effect remain largely unknown. Given their well-known adverse effects, which of the NSAIDs is the best for neurodegenerative disease management remains a matter of debate. Paracetamol is a widely used analgesic/antipyretic drug with low peripheral adverse effects, possibly related to its weak activity as inhibitor of peripheral cyclooxygenase (COX), the main target of NSAIDs. As microglia play an important role in CNS inflammation and pathogenesis of neurodegenerative diseases, we investigate the effect of paracetamol on rat microglial cultures. Although less potent than other NSAIDs, (indomethacin approximately NS-398 > flurbiprofen approximately piroxicam > paracetamol approximately acetylsalicylic acid), paracetamol completely inhibited the synthesis of prostaglandin E(2) (PGE(2)) in lipopolysaccharide-stimulated microglia, when used at concentrations comparable to therapeutic doses. The drug did not affect the expression of the enzymes involved in PGE(2) synthesis, i.e., COX-1, COX-2, and microsomal PGE synthase, or the release of the precursor arachidonic acid (AA). Paracetamol inhibited the conversion of exogenous AA, but not PGH(2), into PGE(2) indicating that the target of the drug is COX activity. Consistently, paracetamol inhibited with similar IC(50) the synthesis of PGF(2alpha) and thromboxane B(2), two other COX metabolites. Finally, none of the NSAIDs affected the productions of nitric oxide and tumor necrosis factor(alpha), two inflammatory mediators released by activated microglia. As paracetamol was reported to inhibit PG synthesis in peripheral macrophages with an IC(50) at least three orders of magnitude higher than in microglia, we suggest that this drug represents a good tool for treating brain inflammation without compromising peripheral PG synthesis.     

A Prion Protein Fragment Primes Type 1 Astrocytes to Proliferation Signals from Microglia

Gliosis is a hallmark of prion disease. A neurotoxic prion peptide (PrP106-126) induces astrocyte proliferation in the presence of microglia. This peptide also directly enhances microglial proliferation in culture. We have investigated this further to understand the method by which factors released by microglia and PrP106-126 work together to enhance astrocyte proliferation. PrP106-126 in the presence of microglia specifically enhanced type 1 astrocyte proliferation but not Type 2. Astrocytes that do not express the prion protein were more sensitive to oxidative stress and the toxicity of cytosine arabinoside. In the presence of cytosine arabinoside, PrP106-126 was toxic to pure astrocyte cultures. Using conditioned medium from microglia we have shown that PrP^c-expressing astrocytes proliferate in response to factors released by microglia stimulated by granulocyte/macrophage colony-stimulating factor. This response is enhanced in the presence of PrP106-126. PrP^c-deficient astrocytes do not show this response. These results suggest that astrocytes are primed by PrP106-126 to respond more to factors released by proliferating microglia. Astrocytes may proliferate in this system to escape entering the cell suicide pathway.     

Fibrillar prion peptide (106-126) and scrapie prion protein hamper phagocytosis in microglia

The inflammatory response in prion diseases is dominated by microglial activation. As macrophages of the central nervous system, the phagocytic capacity of microglia is well recognized, and it is possible that microglia are involved in the removal and processing of amyloid fibrils, thus preventing their harmful effect. We have analyzed the effects of a synthetic peptide of the human prion protein, PrP(106-126), which can form fibrils, and the pathogenic form of prion protein, PrPsc, on phagocytosis in microglia isolated from neonatal rat brain cultures. To some extent, fibrillar PrP(106-126) is internalized and processed. However, both synthetic prion peptide PrP(106-126) in a fibrillar form and pathogenic prion protein PrPsc severely hamper the phagocytic activity as measured by the uptake of beads by microglia. At a concentration that does not induce microglial death, PrP(106-126) reduced the number of beads internalized and altered their cytoplasmic distribution. This effect was not due to decreased binding of beads to the cell surface, nor restricted to specific classes of receptors. Although the PrP(106-126) did not prevent F-actin and Rac1 accumulation at sites of particle engulfment, it appeared to interfere with a later step of the internalization process.     

Hypothermia suppresses inducible nitric oxide synthase and stimulates cyclooxygenase-2 in lipopolysaccharide stimulated BV-2 cells

Hypothermia is neuroprotective, possibly through suppression of microglial activation. We investigated the effects of hypothermia on lipopolysaccharide (LPS) stimulated BV-2 cells. At 37 degrees C, LPS elicited strong increases in inducible nitric oxide synthase (iNOS), nitric oxide (NO), cyclooxygenase-2 (COX-2), tumour necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6), accompanied by translocation of nuclear factor-kappaB (NF-kappaB) to the nucleus. Hypothermia (33 degrees C) caused complete suppression of iNOS and NO, a partial reduction of IL-6 but did not prevent TNF-alpha production or NF-kappaB translocation. In contrast, LPS induced cyclooxygenase-2 (COX-2) to higher levels under hypothermic conditions. These results show that hypothermia selectively suppresses iNOS in microglia.     

Temporal and spatial relationship between the death of PrP-damaged neurones and microglial activation

Previous studies have demonstrated a role for microglia in the neuronal loss that occurs in the transmissible spongiform encephalopathies or prion diseases. In the present studies, the processes that lead to the death of neurones treated with synthetic peptides derived from the prion protein (PrP) were fully activated within 1 h, although neuronal cell death was not seen until 24 h later. Similarly, neurones exposed to PrP peptides for only 1 h activated microglia and a temporal relationship between the production of interleukin-6, an indicator of microglial activation, and microglial killing of PrP-treated neurones was also demonstrated. Activation of microglia and microglia-mediated killing of PrP-treated neurones or scrapie-infected neuroblastoma cells were maximal only when microglia were in direct contact with neurones.     

Comparative study of microglia activation induced by amyloid-beta and prion peptides: role in neurodegeneration

The inflammatory responses in Alzheimer's disease (AD) and prion-related encephalopathies (PRE) are dominated by microglia activation. Several studies have reported that the amyloid-beta (Abeta) peptides, which are associated with AD, and the pathogenic isoform of prion protein (PrPSc) have a crucial role in neuronal death and gliosis that occur in both of these disorders. In this study, we investigate whether Abeta and PrPSc cause microglia activation per se and whether these amyloidogenic peptides differentially affect these immunoeffector cells. In addition, we also determined whether substances released by Abeta- and PrP-activated microglia induce neuronal death. Cultures of rat brain microglia cells were treated with the synthetic peptides Abeta1-40, Abeta1-42 and PrP106-126 for different time periods. The lipopolysaccharide was used as a positive control of microglia activation. Our results show that Abeta1-40 and PrP106-126 caused similar morphological changes in microglia and increased the production of nitric oxide and hydroperoxides. An increase on inducible nitric oxide synthase expression was also observed in microglia treated with Abeta1-40 or PrP106. However, these peptides affected in a different manner the secretion of interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) secretion. In cocultures of microglia-neurons, it was observed that microglia treated with Abeta1-40 or PrP106-126 induced a comparable extent of neuronal death. The neutralizing antibody for IL-6 significantly reduced the neuronal death induced by Abeta- or PrP-activated microglia. Taken together, the data indicate that Abeta and PrP peptides caused microglia activation and differentially affected cytokine secretion. The IL-6 released by reactive microglia caused neuronal injury.     

Abnormal Activation of Microglia Accompanied with Disrupted CX3CR1/CX3CL1 Pathway in the Brains of the Hamsters Infected with Scrapie Agent 263K

Microglial cells are resident mononuclear phagocytes of the central nervous system (CNS). Active proliferation of microglia in the brain has been identified in neurodegenerative disorders, including some kinds of prion disease. However, the detailed regional distribution between microglia and PrP^Sc deposition has not been presented, and investigation of fractalkine signaling which is involved in the regulation of activation of microglia in prion disease is not well documented. In this study, the disease phenomenon of microglial accumulation in the CNS was thoroughly analyzed using a scrapie-infected experimental model. Western blots of microglia-specific markers Iba1 and CD68, immunohistochemical and immunofluorescent assays demonstrated obviously activation of microglia in almost whole brain regions in the infected animals. Under the dynamic analysis on hallmarks of activation of microglia, a time-dependent increase of Iba1 and CD68 was detected, accompanied by accumulation of PrP^Sc and progression of neurodegenerative symptoms. With serial brain sections and double staining of Iba1 and PrP^Sc, we observed that the microglia distributed around PrP^Sc deposits in 263K-infected hamsters’ brains, proposing PrP^Sc phagocytosis. Flow cytometry assays with the single-cell suspensions prepared from the cortical region of the infected brains verified an activation of microglial population. ELISA assays of the cytokines in brain homogenates revealed significant upregulations of interleukin (IL)-1β, IL-6 and TNF-α when infected. Evaluation of fractalkine signaling in the infected hamsters’ brains showed progressively downregulation of CX3CL1 during the incubation. Prion peptide PrP106-126 also disrupted fractalkine and evoked microglial activation in rat primary neuron–glia mixed cultures. Our data here demonstrate an activated status of microglia in CNS tissues of infectious prion disease, possibly through fractalkine signaling deficiency.     

Regulation of IL-1β-Induced Cyclooxygenase-2 Expression by Interactions of Aβ Peptide, Apolipoprotein E and Nitric Oxide in Human Neuroglioma

Alzheimer disease (AD) is characterized by chronic neuroinflammation, which may lead to dysfunction in neuronal circuits. Although reactive microglia are found in association with accumulation of beta amyloid (Aβ) plaques in the AD brain, their contribution to neuronal cell loss remains speculative. A major genetic risk factor for sporadic AD is inheritance of the apolipoprotein (apo) E4 allele, which has been shown to contribute significantly to neurodegeneration in AD. Many studies have documented the ability of Aβ fibrils in vitro to induce microglia to undergo phenotypic activation, which results in the secretion and/or expression of a plethora of free radicals and pro-inflammatory mediators. These mediators, such as reactive nitrogen/oxygen species and IL-1β as well as cyclooxygenase-2 (COX-2) with associated prostaglandin E2 (PGE(2)), are believed to be neurotoxic and to contribute to the underlying cause of AD. We have used the human H4 neuroglioma cells as a model astroglial system to examine the interactions between IL-1β and nitric oxide (NO) as co-stimulators of Aβ(1-40) in enhancing the expression of COX-2 and production of PGE(2) in the presence of recombinant human apolipoprotein E4 (apoE4). Neither Aβ(1-40) nor its reverse sequence analog Aβ(40-1) alone had a significant effect on COX-2 expression and PGE(2) production in the cells. In contrast, after co-incubation with apoE4, Aβ(1-40) increased IL-1β-induced COX-2 expression and PGE(2) production. Aβ(12-28), which binds with high affinity to apoE4, blocked apoE4-mediated effects on Aβ(1-40). Furthermore, (±)-S-Nitroso-N-acetylpenicillamine (SNAP), an agent that releases nitric oxide (NO) in situ, alone did not affect IL-1β-induced COX-2 expression, but increased PGE(2) production only. Addition of Aβ(1-40) preincubated with apoE4 to H4 cells in the presence of SNAP led to an additive IL-1β-induced COX-2 expression and PGE(2) production. These observations indicate that increased PGE(2) resulted from increased nitrosative stress, which is enhanced by apoE4. Thus a molecular understanding of the interactions of apoE4 with Aβ, NO and IL-1β on the regulation of the COX-2/prostaglandin pathway may open new avenues in understanding the mechanism of development of neurodegenerative disease such as AD.     

Thrombin-activated microglia contribute to death of dopaminergic neurons in rat mesencephalic cultures: dual roles of mitogen-activated protein kinase signaling pathways

This study evaluated the role of thrombin-activated microglia in the neurodegeneration of mesencephalic cultures. Immunocytochemical and biochemical evidence indicated that in co-cultures consisting of rat cortical microglia and mesencephalic neurons, thrombin led to nonselective loss of mesencephalic neurons. Accompanying neurodegeneration, microglial activation was obvious, evidenced by expression of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-1beta, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) and by increasing production of TNF-alpha and nitric oxide (NO). In mesencephalic neurons treated with conditioned media (CM) taken from thrombin-activated microglia, the number of dopaminergic neurons was significantly attenuated. The neurotoxicity of the CM was diminished when it was derived from microglia co-treated with thrombin and either an extracellular signal-regulated kinase 1/2 (ERK1/2) pathway inhibitor (PD98059) or a p38-mitogen-activated protein kinase (p38-MAPK) inhibitor (SB203580). Moreover, jun N-terminal kinase (JNK) and p38-MAPK were activated in mesencephalic neurons treated with CM of thrombin-activated microglia. Inhibition of JNK and p38-MAPK rescued the dopaminergic neurons. Collectively, these results indicate that thrombin-activated microglia induce neurodegeneration in cultured mesencephalic neurons and that the MAPKs actively participate in both microglial activation and neurodegeneration. The present data carefully suggest that microglial activation triggered by thrombin may be involved in the neuropathological processes of dopaminergic neuronal cell death that occur in Parkinson's disease.