GPI-anchor signal sequence influences PrPC sorting, shedding and signalling, and impacts on different pathomechanistic aspects of prion disease in mice.

The cellular prion protein (PrPC) is a cell surface glycoprotein attached to the membrane by a glycosylphosphatidylinositol (GPI)-anchor and plays a critical role in transmissible, neurodegenerative and fatal prion diseases. Alterations in membrane attachment influence PrPC-associated signaling, and the development of prion disease, yet our knowledge of the role of the GPI-anchor in localization, processing, and function of PrPC in vivo is limited We exchanged the PrPC GPI-anchor signal sequence of for that of Thy-1 (PrPCGPIThy-1) in cells and mice. We show that this modifies the GPI-anchor composition, which then lacks sialic acid, and that PrPCGPIThy-1 is preferentially localized in axons and is less prone to proteolytic shedding when compared to PrPC. Interestingly, after prion infection, mice expressing PrPCGPIThy-1 show a significant delay to terminal disease, a decrease of microglia/astrocyte activation, and altered MAPK signaling when compared to wild-type mice. Our results are the first to demonstrate in vivo, that the GPI-anchor signal sequence plays a fundamental role in the GPI-anchor composition, dictating the subcellular localization of a given protein and, in the case of PrPC, influencing the development of prion disease.

The phospholipase A2 pathway controls a synaptic cholesterol ester cycle and synapse damage.

The cellular prion protein (PrPC) acts as a scaffold protein that organises signalling complexes. In synaptosomes, the aggregation of PrPC by amyloid-ß (Aß) oligomers attracts and activates cytoplasmic phospholipase A2 (cPLA2), leading to synapse degeneration. The signalling platform is dependent on cholesterol released from cholesterol esters by cholesterol ester hydrolases (CEHs). The activation of cPLA2 requires cholesterol released from cholesterol esters by cholesterol ester hydrolases (CEHs), enzymes dependent upon platelet activating factor (PAF) released by activated cPLA2 This demonstrates a positive feedback system in which activated cPLA2 increased cholesterol concentrations, which in turn facilitated cPLA2 activation. PAF was also required for the incorporation of the tyrosine kinase Fyn and cyclooxygenase (COX)-2 into Aß-PrPC-cPLA2 complexes. As a failure to deactivate signalling complexes can lead to pathology, the mechanisms involved in their dispersal were studied. PAF facilitated the incorporation of acyl-coenzyme A:cholesterol acyltransferase (ACAT)-1 into Aß-PrPC-cPLA2-COX-2-Fyn complexes. The esterification of cholesterol reduced cholesterol concentrations, causing dispersal of Aß-PrPC-cPLA2-COX-2-Fyn complexes and the cessation of signalling. This study identifies PAF as a key mediator regulating the cholesterol ester cycle, activation of cPLA2 and COX-2 within synapses, and synapse damage.

Cholesterol ester hydrolase inhibitors reduce the production of synaptotoxic amyloid-ß oligomers.

The production of amyloid-ß (Aß) is the key factor driving pathogenesis in Alzheimer's disease (AD). Increasing concentrations of Aß within the brain cause synapse degeneration and the dementia that is characteristic of AD. Here the factors that affect the release of disease-relevant forms Aß were studied in a cell model. 7PA2 cells expressing the human amyloid precursor protein released soluble Aß oligomers that caused synapse damage in cultured neurons. Supernatants from 7PA2 cells treated with the cholesterol synthesis inhibitor squalestatin contained similar concentrations of Aß42 to control cells but did not cause synapse damage in neuronal cultures. These supernatants contained reduced concentrations of Aß42 oligomers and increased concentrations of Aß42 monomers. Treatment of 7PA2 cells with platelet-activating factor (PAF) antagonists had similar effects; it reduced concentrations of Aß42 oligomers and increased concentrations of Aß42 monomers in cell supernatants. PAF activated cholesterol ester hydrolases (CEH), enzymes that released cholesterol from stores of cholesterol esters. Inhibition of CEH also reduced concentrations of Aß42 oligomers and increased concentrations of Aß42 monomers in cell supernatants. The Aß monomers produced by treated cells protected neurons against Aß oligomer-induced synapse damage. These studies indicate that pharmacological manipulation of cells can alter the ratio of Aß monomer:oligomer released and consequently their effects on synapses.

Monomeric amyloid-ß reduced amyloid-ß oligomer-induced synapse damage in neuronal cultures.

Alzheimer's disease is a progressive neurodegenerative disease characterized by the accumulation of amyloid-ß (Aß) in the brain. Aß oligomers are believed to cause synapse damage resulting in the memory deficits that are characteristic of this disease. Since the loss of synaptic proteins in the brain correlates closely with the degree of dementia in Alzheimer's disease, the process of Aß-induced synapse damage was investigated in cultured neurons by measuring the loss of synaptic proteins. Soluble Aß oligomers, derived from Alzheimer's-affected brains, caused the loss of cysteine string protein and synaptophysin from neurons. When applied to synaptosomes Aß oligomers increased cholesterol concentrations and caused aberrant activation of cytoplasmic phospholipase A2 (cPLA2). In contrast, Aß monomer preparations did not affect cholesterol concentrations or activate synaptic cPLA2, nor did they damage synapses. The Aß oligomer-induced aggregation of cellular prion proteins (PrPC) at synapses triggered the activation of cPLA2 that leads to synapse degeneration. Critically, Aß monomer preparations did not cause the aggregation of PrPC; rather they reduced the Aß oligomer-induced aggregation of PrPC. The presence of Aß monomer preparations also inhibited the Aß oligomer-induced increase in cholesterol concentrations and activation of cPLA2 in synaptosomes and protected neurons against the Aß oligomer-induced synapse damage. These results support the hypothesis that Aß monomers are neuroprotective. We hypothesise that synapse damage may result from a pathological Aß monomer:oligomer ratio rather than the total concentrations of Aß within the brain.

Valproic acid and its congener propylisopropylacetic acid reduced the amount of soluble amyloid-ß oligomers released from 7PA2 cells.

The amyloid hypothesis of Alzheimer's disease suggests that synaptic degeneration and pathology is caused by the accumulation of amyloid-ß (Aß) peptides derived from the amyloid precursor protein (APP). Subsequently, soluble Aß oligomers cause the loss of synaptic proteins from neurons, a histopathological feature of Alzheimer's disease that correlates with the degree of dementia. In this study, the production of toxic forms of Aß was examined in vitro using 7PA2 cells stably transfected with human APP. We show that conditioned media from 7PA2 cells containing Aß oligomers caused synapse degeneration as measured by the loss of synaptic proteins, including synaptophysin and cysteine-string protein, from cultured neurons. Critically, conditioned media from 7PA2 cells treated with valproic acid (2-propylpentanoic acid (VPA)) or propylisopropylacetic acid (PIA) did not cause synapse damage. Treatment with VPA or PIA did not significantly affect total Aß42 concentrations; rather these drugs selectively reduced the concentrations of Aß42 oligomers in conditioned media. In contrast, treatment significantly increased the concentrations of Aß42 monomers in conditioned media. VPA or PIA treatment reduced the concentrations of APP within lipid rafts, membrane compartments associated with Aß production. These effects of VPA and PIA were reversed by the addition of platelet-activating factor, a bioactive phospholipid produced following activation of phospholipase A2, an enzyme sensitive to VPA and PIA. Collectively these data suggest that VPA and PIA reduce Aß oligomers through inhibition of phospholipase A2 and suggest a novel therapeutic approach to Alzheimer's treatment.

Breaking the Cycle, Cholesterol Cycling, and Synapse Damage in Response to Amyloid-ß.

Soluble amyloid-ß (Aß) oligomers, a key driver of pathogenesis in Alzheimer disease, bind to cellular prion proteins (PrPC) expressed on synaptosomes resulting in increased cholesterol concentrations, movement of cytoplasmic phospholipase A2 (cPLA2) to lipid rafts and activation of cPLA2. The formation of Aß-PrPC-cPLA2 complexes was controlled by the cholesterol ester cycle. Thus, Aß activated cholesterol ester hydrolases which released cholesterol from stores of cholesterol esters; the increased cholesterol concentrations stabilised Aß-PrPC-cPLA2 complexes. Conversely, cholesterol esterification reduced cholesterol concentrations causing the dispersal of Aß-PrPC-cPLA2. In cultured neurons, the cholesterol ester cycle regulated Aß-induced synapse damage; inhibition of cholesterol ester hydrolases protected neurons, whereas inhibition of cholesterol esterification increased the Aß-induced synapse damage. Here, I speculate that a failure to deactivate signalling pathways can lead to pathology. Consequently, the esterification of cholesterol is a key factor in the dispersal of Aß-induced signalling platforms and synapse degeneration.

The cholesterol ester cycle regulates signalling complexes and synapse damage caused by amyloid-ß.

Cholesterol is required for the formation and function of some signalling platforms. In synaptosomes, amyloid-ß (Aß) oligomers, the causative agent in Alzheimer's disease, bind to cellular prion proteins (PrPC) resulting in increased cholesterol concentrations, translocation of cytoplasmic phospholipase A2 (cPLA2, also known as PLA2G4A) to lipid rafts, and activation of cPLA2 The formation of Aß-PrPC complexes is controlled by the cholesterol ester cycle. In this study, Aß activated cholesterol ester hydrolases, which released cholesterol from stores of cholesterol esters and stabilised Aß-PrPC complexes, resulting in activated cPLA2 Conversely, cholesterol esterification reduced cholesterol concentrations causing the dispersal of Aß-PrPC complexes. In cultured neurons, the cholesterol ester cycle regulated Aß-induced synapse damage; cholesterol ester hydrolase inhibitors protected neurons, while inhibition of cholesterol esterification significantly increased Aß-induced synapse damage. An understanding of the molecular mechanisms involved in the dispersal of signalling complexes is important as failure to deactivate signalling pathways can lead to pathology. This study demonstrates that esterification of cholesterol is a key factor in the dispersal of Aß-induced signalling platforms involved in the activation of cPLA2 and synapse degeneration.

Sialylated glycosylphosphatidylinositols suppress the production of toxic amyloid-ß oligomers.

The production of amyloid-ß (Aß) is a key factor driving pathogenesis in Alzheimer's disease (AD). Increasing concentrations of soluble Aß oligomers within the brain lead to synapse degeneration and the progressive dementia characteristic of AD. Since Aß exists in both disease-relevant (toxic) and non-toxic forms, the factors that affected the release of toxic Aß were studied in a cell model. 7PA2 cells expressing the human amyloid precursor protein released Aß oligomers that caused synapse damage when incubated with cultured neurones. These Aß oligomers had similar potency to soluble Aß oligomers derived from the brains of Alzheimer's patients. Although the conditioned media from 7PA2 cells treated with the cellular prion protein (PrPC) contained Aß, it did not cause synapse damage. The loss of toxicity was associated with a reduction in Aß oligomers and an increase in Aß monomers. The suppression of toxic Aß release was dependent on the glycosylphosphatidylinositol (GPI) anchor attached to PrPC, and treatment of cells with specific GPIs alone reduced the production of toxic Aß. The efficacy of GPIs was structure-dependent and the presence of sialic acid was critical. The conditioned medium from GPI-treated cells protected neurones against Aß oligomer-induced synapse damage; neuroprotection was mediated by Aß monomers. These studies support the hypothesis that the ratio of Aß monomers to Aß oligomers is a critical factor that regulates synapse damage.

Sialic Acid within the Glycosylphosphatidylinositol Anchor Targets the Cellular Prion Protein to Synapses.

Although the cellular prion protein (PrP(C)) is concentrated at synapses, the factors that target PrP(C) to synapses are not understood. Here we demonstrate that exogenous PrP(C) was rapidly targeted to synapses in recipient neurons derived from Prnp knock-out((0/0)) mice. The targeting of PrP(C) to synapses was dependent upon both neuronal cholesterol concentrations and the lipid and glycan composition of its glycosylphosphatidylinositol (GPI) anchor. Thus, the removal of either an acyl chain or sialic acid from the GPI anchor reduced the targeting of PrP(C) to synapses. Isolated GPIs (derived from PrP(C)) were also targeted to synapses, as was IgG conjugated to these GPIs. The removal of sialic acid from GPIs prevented the targeting of either the isolated GPIs or the IgG-GPI conjugate to synapses. Competition studies showed that pretreatment with sialylated GPIs prevented the targeting of PrP(C) to synapses. These results are consistent with the hypothesis that the sialylated GPI anchor attached to PrP(C) acts as a synapse homing signal.

Glycosylphosphatidylinositols: More than just an anchor?

There is increasing interest in the role of glycosylphosphatidylinositol (GPI) anchors that attach some proteins to cell membranes. Far from being biologically inert, GPIs influence the targeting, intracellular trafficking and function of the attached protein. Our recent paper demonstrated the role of sialic acid on the GPI of the cellular prion protein (PrP(C)). The "prion diseases" arise following the conversion of PrP(C) to a disease-associated isoform called PrP(Sc) or "prion". Our paper showed that desialylated PrP(C) inhibited PrP(Sc) formation. Aggregated PrP(Sc) creates a signaling platform in the cell membrane incorporating and activating cytoplasmic phospholipase A2 (cPLA2), an enzyme that regulates PrP(C) trafficking and hence PrP(Sc) formation. The presence of desialylated PrP(C) caused the dissociation of cPLA2 from PrP-containing platforms, reduced the activation of cPLA2 and inhibited PrP(Sc) production. We concluded that sialic acid contained within the GPI attached to PrP(C) modifies local membrane microenvironments that are important in PrP-mediated cell signaling and PrP(Sc) formation.

Does the tail wag the dog? How the structure of a glycosylphosphatidylinositol anchor affects prion formation.

There is increasing interest in the role of the glycosylphosphatidylinositol (GPI) anchor attached to the cellular prion protein (PrP(C)). Since GPI anchors can alter protein targeting, trafficking and cell signaling, our recent study examined how the structure of the GPI anchor affected prion formation. PrP(C) containing a GPI anchor from which the sialic acid had been removed (desialylated PrP(C)) was not converted to PrP(Sc) in prion-infected neuronal cell lines and in scrapie-infected primary cortical neurons. In uninfected neurons desialylated PrP(C) was associated with greater concentrations of gangliosides and cholesterol than PrP(C). In addition, the targeting of desialylated PrP(C) to lipid rafts showed greater resistance to cholesterol depletion than PrP(C). The presence of desialylated PrP(C) caused the dissociation of cytoplasmic phospholipase A2 (cPLA2) from PrP-containing lipid rafts, reduced the activation of cPLA2 and inhibited PrP(Sc) production. We conclude that the sialic acid moiety of the GPI attached to PrP(C) modifies local membrane microenvironments that are important in PrP-mediated cell signaling and PrP(Sc) formation.

An in vitro model for synaptic loss in neurodegenerative diseases suggests a neuroprotective role for valproic acid via inhibition of cPLA2 dependent signalling.

Many neurodegenerative diseases present the loss of synapses as a common pathological feature. Here we have employed an in vitro model for synaptic loss to investigate the molecular mechanism of a therapeutic treatment, valproic acid (VPA). We show that amyloid-ß (Aß), isolated from patient tissue and thought to be the causative agent of Alzheimer's disease, caused the loss of synaptic proteins including synaptophysin, synapsin-1 and cysteine-string protein from cultured mouse neurons. Aß-induced synapse damage was reduced by pre-treatment with physiologically relevant concentrations of VPA (10 µM) and a structural variant propylisopropylacetic acid (PIA). These drugs also reduced synaptic damage induced by other neurodegenerative-associated proteins α-synuclein, linked to Lewy body dementia and Parkinson's disease, and the prion-derived peptide PrP82-146. Consistent with these effects, synaptic vesicle recycling was also inhibited by these proteins and protected by VPA and PIA. We show a mechanism for this damage through aberrant activation of cytoplasmic phospholipase A2 (cPLA2) that is reduced by both drugs. Furthermore, Aß-dependent cPLA2 activation correlates with its accumulation in lipid rafts, and is likely to be caused by elevated cholesterol (stabilising rafts) and decreased cholesterol ester levels, and this mechanism is reduced by VPA and PIA. Such observations suggest that VPA and PIA may provide protection against synaptic damage that occurs during Alzheimer's and Parkinson's and prion diseases.

Glimepiride protects neurons against amyloid-ß-induced synapse damage.

Alzheimer's disease is associated with the accumulation within the brain of amyloid-ß (Aß) peptides that damage synapses and affect memory acquisition. This process can be modelled by observing the effects of Aß on synapses in cultured neurons. The addition of picomolar concentrations of soluble Aß derived from brain extracts triggered the loss of synaptic proteins including synaptophysin, synapsin-1 and cysteine string protein from cultured neurons. Glimepiride, a sulphonylurea used for the treatment of diabetes, protected neurons against synapse damage induced by Aß. The protective effects of glimepiride were multi-faceted. Glimepiride treatment was associated with altered synaptic membranes including the loss of specific glycosylphosphatidylinositol (GPI)-anchored proteins including the cellular prion protein (PrP(C)) that acts as a receptor for Aß42, increased synaptic gangliosides and altered cell signalling. More specifically, glimepiride reduced the Aß-induced increase in cholesterol and the Aß-induced activation of cytoplasmic phospholipase A2 (cPLA2) in synapses that occurred within cholesterol-dense membrane rafts. Aß42 binding to glimepiride-treated neurons was not targeted to membrane rafts and less Aß42 accumulated within synapses. These studies indicate that glimepiride modified the membrane micro-environments in which Aß-induced signalling leads to synapse damage. In addition, soluble PrP(C), released from neurons by glimepiride, neutralised Aß-induced synapse damage. Such observations raise the possibility that glimepiride may reduce synapse damage and hence delay the progression of cognitive decline in Alzheimer's disease.

Sialic Acid on the Glycosylphosphatidylinositol Anchor Regulates PrP-mediated Cell Signaling and Prion Formation.

The prion diseases occur following the conversion of the cellular prion protein (PrP(C)) into disease-related isoforms (PrP(Sc)). In this study, the role of the glycosylphosphatidylinositol (GPI) anchor attached to PrP(C) in prion formation was examined using a cell painting technique. PrP(Sc) formation in two prion-infected neuronal cell lines (ScGT1 and ScN2a cells) and in scrapie-infected primary cortical neurons was increased following the introduction of PrP(C). In contrast, PrP(C) containing a GPI anchor from which the sialic acid had been removed (desialylated PrP(C)) was not converted to PrP(Sc). Furthermore, the presence of desialylated PrP(C) inhibited the production of PrP(Sc) within prion-infected cortical neurons and ScGT1 and ScN2a cells. The membrane rafts surrounding desialylated PrP(C) contained greater amounts of sialylated gangliosides and cholesterol than membrane rafts surrounding PrP(C). Desialylated PrP(C) was less sensitive to cholesterol depletion than PrP(C) and was not released from cells by treatment with glimepiride. The presence of desialylated PrP(C) in neurons caused the dissociation of cytoplasmic phospholipase A2 from PrP-containing membrane rafts and reduced the activation of cytoplasmic phospholipase A2. These findings show that the sialic acid moiety of the GPI attached to PrP(C) modifies local membrane microenvironments that are important in PrP-mediated cell signaling and PrP(Sc) formation. These results suggest that pharmacological modification of GPI glycosylation might constitute a novel therapeutic approach to prion diseases.

cAMP-Inhibits Cytoplasmic Phospholipase A2 and Protects Neurons against Amyloid-ß-Induced Synapse Damage.

A key event in Alzheimer's disease (AD) is the production of amyloid-ß (Aß) peptides and the loss of synapses. In cultured neurons Aß triggered synapse damage as measured by the loss of synaptic proteins. α-synuclein (αSN), aggregates of which accumulate in Parkinson's disease, also caused synapse damage. Synapse damage was associated with activation of cytoplasmic phospholipase A2 (cPLA2), an enzyme that regulates synapse function and structure, and the production of prostaglandin (PG) E2. In synaptosomes PGE2 increased concentrations of cyclic adenosine monophosphate (cAMP) which suppressed the activation of cPLA2 demonstrating an inhibitory feedback system. Thus, Aß/αSN-induced activated cPLA2 produces PGE2 which increases cAMP which in turn suppresses cPLA2 and, hence, its own production. Neurons pre-treated with pentoxifylline and caffeine (broad spectrum phosphodiesterase (PDE) inhibitors) or the PDE4 specific inhibitor rolipram significantly increased the Aß/αSN-induced increase in cAMP and consequently protected neurons against synapse damage. The addition of cAMP analogues also inhibited cPLA2 and protected neurons against synapse damage. These results suggest that drugs that inhibit Aß-induced activation of cPLA2 and cross the blood-brain barrier may reduce synapse damage in AD.

Monoacylated Cellular Prion Proteins Reduce Amyloid-ß-Induced Activation of Cytoplasmic Phospholipase A2 and Synapse Damage.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by the accumulation of amyloid-ß (Aß) and the loss of synapses. Aggregation of the cellular prion protein (PrPC) by Aß oligomers induced synapse damage in cultured neurons. PrPC is attached to membranes via a glycosylphosphatidylinositol (GPI) anchor, the composition of which affects protein targeting and cell signaling. Monoacylated PrPC incorporated into neurons bound "natural Aß", sequestering Aß outside lipid rafts and preventing its accumulation at synapses. The presence of monoacylated PrPC reduced the Aß-induced activation of cytoplasmic phospholipase A2 (cPLA2) and Aß-induced synapse damage. This protective effect was stimulus specific, as treated neurons remained sensitive to α-synuclein, a protein associated with synapse damage in Parkinson's disease. In synaptosomes, the aggregation of PrPC by Aß oligomers triggered the formation of a signaling complex containing the cPLA2.a process, disrupted by monoacylated PrPC. We propose that monoacylated PrPC acts as a molecular sponge, binding Aß oligomers at the neuronal perikarya without activating cPLA2 or triggering synapse damage.

α-Synuclein-induced synapse damage in cultured neurons is mediated by cholesterol-sensitive activation of cytoplasmic phospholipase A2.

The accumulation of aggregated forms of the α-synuclein (αSN) is associated with the pathogenesis of Parkinson's disease (PD) and Dementia with Lewy Bodies. The loss of synapses is an important event in the pathogenesis of these diseases. Here we show that aggregated recombinant human αSN, but not ßSN, triggered synapse damage in cultured neurons as measured by the loss of synaptic proteins. Pre-treatment with the selective cytoplasmic phospholipase A2 (cPLA2) inhibitors AACOCF3 and MAFP protected neurons against αSN-induced synapse damage. Synapse damage was associated with the αSN-induced activation of synaptic cPLA2 and the production of prostaglandin E2. The activation of cPLA2 is the first step in the generation of platelet-activating factor (PAF) and PAF receptor antagonists (ginkgolide B or Hexa-PAF) also protect neurons against αSN-induced synapse damage. αSN-induced synapse damage was also reduced in neurons pre-treated with the cholesterol synthesis inhibitor (squalestatin). These results are consistent with the hypothesis that αSN triggered synapse damage via hyperactivation of cPLA2. They also indicate that αSN-induced activation of cPLA2 is influenced by the cholesterol content of membranes. Inhibitors of this pathway that can cross the blood brain barrier may protect against the synapse damage seen during PD.

Glimepiride reduces CD14 expression and cytokine secretion from macrophages.

BACKGROUND: Activated microglia are associated with deposits of aggregated proteins within the brains of patients with Alzheimer's disease (AD), Parkinson's disease (PD) and prion diseases. Since the cytokines secreted from activated microglia are thought to contribute to the pathogenesis of these neurodegenerative diseases, compounds that suppress cytokine production have been identified as potential therapeutic targets. CD14 is a glycosylphosphatidylinositol (GPI)- anchored protein that is part of a receptor complex that mediates microglial responses to peptides that accumulate in prion disease (PrP82-146), AD (amyloid-ß (Aß)42) and PD (α-synuclein (αSN)). As some GPI-anchored proteins are released from cells by treatment with glimepiride, a sulphonylurea used for the treatment of diabetes, the effects of glimepiride upon CD14 expression and cytokine production from cultured macrophages were studied. METHODS: RAW 264 cells and microglial cells were treated with glimepiride or phosphatidylinositol (PI)-phospholipase C (PLC) and the expression of cell receptors was analysed by ELISA and immunoblot. Treated cells were subsequently incubated with Aß42, αSN, PrP82-146 or lipopolysaccharide (LPS) and the amounts of Toll-like receptor (TLR)-4, tumour necrosis factor (TNF), interleukin (IL)-1 and IL-6 measured. RESULTS: Glimepiride released CD14 from RAW 264 cells and microglial cells. Pre-treatment with glimepiride significantly reduced TNF, IL-1 and IL-6 secretion from RAW 264 and microglial cells incubated with LPS, Aß42, αSN and PrP82-146. Glimepiride also reduced the LPS, Aß42, αSN and PrP82-146-induced translocation of TLR-4 into membrane rafts that is associated with cell activation. These effects of glimepiride were also seen after digestion of RAW 264 cells with PI-phospholipase C (PLC). In addition, the effects of glimepiride were blocked by pharmacological inhibition of GPI-PLC. The cytokine production was CD14-dependent; it was reduced in microglia from CD14 knockout mice and was blocked by antiserum to CD14. CONCLUSIONS: RAW 264 and microglial cell responses to Aß1-42, αSN, PrP82-146 and LPS are dependent upon CD14 expression. Glimepiride induced the shedding of CD14 from cells by activation of GPI-PLC and consequently reduced cytokine production in response to Aß42, αSN, PrP82-146 and LPS. These results suggest that glimepiride acts as a novel anti-inflammatory agent that could modify the progression of neurodegenerative diseases.