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P3-355: Oligomeric beta-amyloid peptide stimulates a unique microglial activation phenotype
T626 P3-355
Poster Presentations P3: OLIGOMERIC BETA-AMYLOID PEPTIDE STIMULATES A UNIQUE MICROGLIAL ACTIVATION PHENOTYPE
Colin K. Combs, Angela Floden, Cindy Sondag, Universitty of North Dakota, Grand Forks, ND, USA. Contact e-mail: [email protected] Background: Fibrillar beta amyloid peptides are a major component of the senile plaques present in Alzheimer’s disease brain. Although this conformation has the ability to stimulate microglial activation in vitro and in vivo, recent data suggests that a prefibrillar, oligomeric species may also serve as a microglial stimulus. Methods: In order to compare microglial stimulatory properties of fibrillar and oligomeric beta amyloid peptides we have utilized primary mouse microglia cultures. Results: Although stimulation with either conformation was sufficient to induce a tyrosine kinase-based signaling response in microglia, the resultant cytokine secretory profile was unique for each peptide form. In addition, oligomeric peptide-mediated loss of primary neuron cultures was dependent upon co-culture with microglia in contrast to fibrils which were potently toxic to neuron cultures alone. In order validate the culture findings, oligomeric peptide was intracerebroventricularly infused into six month old male C57BL/6 mice to assess whether a similar increase in microglial activation and neuron loss would occur. Infusion of oligomeric peptide induced a preferential loss of presynaptic versus postsynaptic protein markers. This correlated with increased immunoreactivity for reactive microglia and astrocytes as well as increased active levels of the tyrosine kinase, Lyn. Conclusions: These data suggest a role for beta amyloid oligomers in the proinflammatory activation of microglia that is distinct from that of fibrils and suggest a role in neuron loss and synaptic dysfunction during disease. P3-356
LIPOXYGENASES IN THE EARLY STAGE OF AMYLOID-BETA PEPTIDES TOXICITY AND IN MEMORY IMPAIRMENT: THE MODULATORY ROLE OF INFLAMMATION
Grzegorz A. Czapski, Magdalena Cakala, Joanna B. Strosznajder, Medical Research Centre, Warsaw, Poland. Contact e-mail: [email protected] Background: Amyloid precursor protein (APP) is processed into soluble APP and amyloid-beta (AB) peptides. Oligomers of AB liberated in high amount in Alzheimer’s Disease (AD) lead to synaptosis and memory disturbances. However, the molecular mechanism of AB toxicity and AD is not fully understood. Methods: We have investigated the role of A peptides-evoked alterations of lipoxygenases (LOX) expression and activity in PC12 cells and the role of lipoxygenases in AB1-42-evoked memory impairment in C57BL6 mice. Results: Using PC12 control cells and cells transfected with APP gene we have demonstrated that AB enhanced NO synthase activity and that NO by affecting GMP/PKG was responsible for activation of phospholipase A2 (PLA2) and for enhancement of arachidonic acid metabolism by cyclooxygenases (COX) and LOX. This AB-induced metabolic pathway was decreased by inhibitors of gamma-secretase, NOS and cPLA2. Inhibitor of 12-LOX (baicalein) protected also the cells against NFkappa B translocation and death. AB enhanced 12-LOX mRNA level, protein level and activity. In vivo studies indicated that AB enhanced 12-LOX protein level in hippocampus and decreased locomotion and memory in mice C57BL6 injected (iv) with AB alone or together with lipopolysaccharide (ip). Baicalein protected mice against memory deficit and locomotory disturbances. Our results suggested also the important role of 5-LOX. Conclusions: The 12-LOX inhibitor protects neurons against death and mice against memory deficit and locomotory disturbances. Supported by: MS&HE scientific network 28/E-32/SN0053/2007.
P3-357
CHANGES IN EXPRESSION PATTERNS OF PGE2 SYNTHASES IN ALZHEIMER’S DISEASE BRAINS
Sylvain Dore, Uzma A. Chaudhry, Johns Hopkins University, Baltimore, MD, USA. Contact e-mail: [email protected] Background: The proinflammatory prostaglandin PGE2 has been found to be elevated in cerebrospinal fluid early in Alzheimer disease (AD). Interestingly, nonsteroidal anti-inflammatory drugs (NSAIDs) are able to reduce PGE2 production and have been shown to reduce the risk of AD. Nevertheless, in clinical studies, cyclooxygenase (COX)-2 inhibitors and NSAIDs have not been effective in slowing or reversing AD progression or cognitive decline. Because COX-2 inhibition could block the generation of other arachidonic acid metabolites, such as PGD2, PGF2␣, PGI2, and thromboxanes, inhibition of a selective PGE synthase could be an alternative. Three PGE synthases have been identified: cytosolic PGES (cPGES), microsomal PGES-1 (mPGES-1) and mPGES-2, and here we studied their expression patterns in AD. Methods: Middle frontal gyrus tissue from AD and age-matched control brains was analyzed by Western blot, immunofluorescence, and immunohistochemistry with specific antibodies. Results: We first reported that mPGES1 was detected by immunofluorescence in neurons, microglia, astrocytes, and endothelial cells of middle frontal gyrus tissue from AD patients and age-matched controls. Western blot analysis showed that mPGES-1 expression was significantly elevated in AD tissue. Here, we investigated the cPGES, a ubiquitous, constitutively expressed, immediate source of PGE2. Immunofluorescence analysis of control brains showed that cPGES colocalizes with markers for microglia, neurons, and endothelium, but not with those for astrocytes or smooth muscle cells. Furthermore, immunohistochemical staining for cPGES appeared to be more intense in the pyramidal neurons of control brain sections than in those of AD brain sections. Work is ongoing to characterize mPGES-2 in AD. Conclusions: Taken together, these findings suggest that cPGES and mPGES-1 have unique patterns of distribution in the human brain. Diseaserelated changes in expression levels make these enzymes potential pharmacological targets to modulate the various cellular actions of PGE2 and to potentially reduce the inflammatory process involve in the etiopathology of AD. P3-358
TOLL-LIKE RECEPTOR 4-DEPENDENT UPREGULATION OF CYTOKINES IN A TRANSGENIC MOUSE MODEL OF ALZHEIMER’S DISEASE
Ken-ichiro Fukuchi1, Jing-Ji Jin1, Hong-Duck Kim1, J. Adam Maxwell1, Ling Li2, 1University of Illinois College of Medicine at Peoria, Peoria, IL, USA; 2University of Alabama at Birmingham, Birmingham, AL, USA. Contact e-mail: [email protected] Background: A deposits in the brains of patients with Alzheimer’s disease (AD) are closely associated with innate immune responses such as activated microglia and increased cytokines. Accumulating evidence supports the hypothesis that innate immune/inflammatory responses play a pivotal role in the pathogenesis of AD: either beneficial or harmful effects on the AD progression. The molecular mechanisms, however, by which the innate immune system modulates the AD progression are not well understood. Toll-like receptors (TLRs) are first-line molecules for initiating the innate immune responses. Activated microglia by exogenous and endogenous TLR ligands respond to insults including pathogens and damaged host cells by secreting chemokines and cytokines, and express the co-stimulatory molecules needed for protective immune responses and efficient clearance of damaged tissues. Here, we investigated the roles of TLR4 in A-induced upregulation of cytokines and chemokines and in A-induced activation of microglia and astrocytes. Methods: We previously demonstrated that an AD mouse model (Mo/Hu APPswe PS1dE9 mice) homozygous for a destructive mutation of TLR4 (TlrLps-d/TlrLps-d) had increases in diffuse and fibrillar A deposits as well as buffer-soluble and insoluble A in the brain as compared with a TLR4 wild-type AD mouse model (Tahara et al. Brain 129: 3006-3019, 2006). Using the same model, levels of
Poster Presentations P3: OLIGOMERIC BETA-AMYLOID PEPTIDE STIMULATES A UNIQUE MICROGLIAL ACTIVATION PHENOTYPE
Colin K. Combs, Angela Floden, Cindy Sondag, Universitty of North Dakota, Grand Forks, ND, USA. Contact e-mail: [email protected] Background: Fibrillar beta amyloid peptides are a major component of the senile plaques present in Alzheimer’s disease brain. Although this conformation has the ability to stimulate microglial activation in vitro and in vivo, recent data suggests that a prefibrillar, oligomeric species may also serve as a microglial stimulus. Methods: In order to compare microglial stimulatory properties of fibrillar and oligomeric beta amyloid peptides we have utilized primary mouse microglia cultures. Results: Although stimulation with either conformation was sufficient to induce a tyrosine kinase-based signaling response in microglia, the resultant cytokine secretory profile was unique for each peptide form. In addition, oligomeric peptide-mediated loss of primary neuron cultures was dependent upon co-culture with microglia in contrast to fibrils which were potently toxic to neuron cultures alone. In order validate the culture findings, oligomeric peptide was intracerebroventricularly infused into six month old male C57BL/6 mice to assess whether a similar increase in microglial activation and neuron loss would occur. Infusion of oligomeric peptide induced a preferential loss of presynaptic versus postsynaptic protein markers. This correlated with increased immunoreactivity for reactive microglia and astrocytes as well as increased active levels of the tyrosine kinase, Lyn. Conclusions: These data suggest a role for beta amyloid oligomers in the proinflammatory activation of microglia that is distinct from that of fibrils and suggest a role in neuron loss and synaptic dysfunction during disease. P3-356
LIPOXYGENASES IN THE EARLY STAGE OF AMYLOID-BETA PEPTIDES TOXICITY AND IN MEMORY IMPAIRMENT: THE MODULATORY ROLE OF INFLAMMATION
Grzegorz A. Czapski, Magdalena Cakala, Joanna B. Strosznajder, Medical Research Centre, Warsaw, Poland. Contact e-mail: [email protected] Background: Amyloid precursor protein (APP) is processed into soluble APP and amyloid-beta (AB) peptides. Oligomers of AB liberated in high amount in Alzheimer’s Disease (AD) lead to synaptosis and memory disturbances. However, the molecular mechanism of AB toxicity and AD is not fully understood. Methods: We have investigated the role of A peptides-evoked alterations of lipoxygenases (LOX) expression and activity in PC12 cells and the role of lipoxygenases in AB1-42-evoked memory impairment in C57BL6 mice. Results: Using PC12 control cells and cells transfected with APP gene we have demonstrated that AB enhanced NO synthase activity and that NO by affecting GMP/PKG was responsible for activation of phospholipase A2 (PLA2) and for enhancement of arachidonic acid metabolism by cyclooxygenases (COX) and LOX. This AB-induced metabolic pathway was decreased by inhibitors of gamma-secretase, NOS and cPLA2. Inhibitor of 12-LOX (baicalein) protected also the cells against NFkappa B translocation and death. AB enhanced 12-LOX mRNA level, protein level and activity. In vivo studies indicated that AB enhanced 12-LOX protein level in hippocampus and decreased locomotion and memory in mice C57BL6 injected (iv) with AB alone or together with lipopolysaccharide (ip). Baicalein protected mice against memory deficit and locomotory disturbances. Our results suggested also the important role of 5-LOX. Conclusions: The 12-LOX inhibitor protects neurons against death and mice against memory deficit and locomotory disturbances. Supported by: MS&HE scientific network 28/E-32/SN0053/2007.
P3-357
CHANGES IN EXPRESSION PATTERNS OF PGE2 SYNTHASES IN ALZHEIMER’S DISEASE BRAINS
Sylvain Dore, Uzma A. Chaudhry, Johns Hopkins University, Baltimore, MD, USA. Contact e-mail: [email protected] Background: The proinflammatory prostaglandin PGE2 has been found to be elevated in cerebrospinal fluid early in Alzheimer disease (AD). Interestingly, nonsteroidal anti-inflammatory drugs (NSAIDs) are able to reduce PGE2 production and have been shown to reduce the risk of AD. Nevertheless, in clinical studies, cyclooxygenase (COX)-2 inhibitors and NSAIDs have not been effective in slowing or reversing AD progression or cognitive decline. Because COX-2 inhibition could block the generation of other arachidonic acid metabolites, such as PGD2, PGF2␣, PGI2, and thromboxanes, inhibition of a selective PGE synthase could be an alternative. Three PGE synthases have been identified: cytosolic PGES (cPGES), microsomal PGES-1 (mPGES-1) and mPGES-2, and here we studied their expression patterns in AD. Methods: Middle frontal gyrus tissue from AD and age-matched control brains was analyzed by Western blot, immunofluorescence, and immunohistochemistry with specific antibodies. Results: We first reported that mPGES1 was detected by immunofluorescence in neurons, microglia, astrocytes, and endothelial cells of middle frontal gyrus tissue from AD patients and age-matched controls. Western blot analysis showed that mPGES-1 expression was significantly elevated in AD tissue. Here, we investigated the cPGES, a ubiquitous, constitutively expressed, immediate source of PGE2. Immunofluorescence analysis of control brains showed that cPGES colocalizes with markers for microglia, neurons, and endothelium, but not with those for astrocytes or smooth muscle cells. Furthermore, immunohistochemical staining for cPGES appeared to be more intense in the pyramidal neurons of control brain sections than in those of AD brain sections. Work is ongoing to characterize mPGES-2 in AD. Conclusions: Taken together, these findings suggest that cPGES and mPGES-1 have unique patterns of distribution in the human brain. Diseaserelated changes in expression levels make these enzymes potential pharmacological targets to modulate the various cellular actions of PGE2 and to potentially reduce the inflammatory process involve in the etiopathology of AD. P3-358
TOLL-LIKE RECEPTOR 4-DEPENDENT UPREGULATION OF CYTOKINES IN A TRANSGENIC MOUSE MODEL OF ALZHEIMER’S DISEASE
Ken-ichiro Fukuchi1, Jing-Ji Jin1, Hong-Duck Kim1, J. Adam Maxwell1, Ling Li2, 1University of Illinois College of Medicine at Peoria, Peoria, IL, USA; 2University of Alabama at Birmingham, Birmingham, AL, USA. Contact e-mail: [email protected] Background: A deposits in the brains of patients with Alzheimer’s disease (AD) are closely associated with innate immune responses such as activated microglia and increased cytokines. Accumulating evidence supports the hypothesis that innate immune/inflammatory responses play a pivotal role in the pathogenesis of AD: either beneficial or harmful effects on the AD progression. The molecular mechanisms, however, by which the innate immune system modulates the AD progression are not well understood. Toll-like receptors (TLRs) are first-line molecules for initiating the innate immune responses. Activated microglia by exogenous and endogenous TLR ligands respond to insults including pathogens and damaged host cells by secreting chemokines and cytokines, and express the co-stimulatory molecules needed for protective immune responses and efficient clearance of damaged tissues. Here, we investigated the roles of TLR4 in A-induced upregulation of cytokines and chemokines and in A-induced activation of microglia and astrocytes. Methods: We previously demonstrated that an AD mouse model (Mo/Hu APPswe PS1dE9 mice) homozygous for a destructive mutation of TLR4 (TlrLps-d/TlrLps-d) had increases in diffuse and fibrillar A deposits as well as buffer-soluble and insoluble A in the brain as compared with a TLR4 wild-type AD mouse model (Tahara et al. Brain 129: 3006-3019, 2006). Using the same model, levels of