MyD88 is dispensable for cerebral amyloid pathology and neuroinflammation in PSAPP transgenic mice

P302

Poster Presentations: P2

Background: CHF5074, nonsteroidal anti-inflammatory derivative with g -secretase modulatory activity has been shown to inhibit brain plaque deposition and to attenuate or reverse memory deficit in different transgenic mouse models of Alzheimer disease (AD). The recent discovery of modulation Rho-GTPase-dependent signaling suggests that the range of biological actions exerted by this drug may be wider than expected. Since neuroinflammation is considered a major pathogenetic mechanism in AD, we investigated the possible regulation of microglia activation by CHF5074 in vivo and in vitro. Methods: The Tg2576 transgenic mouse carrying a transgene coding for the 695-amino acid isoform of human APP derived from a large Swedish family with early-onset AD was used for in vivo study. CHF5074 60 mg/kg was administered for 2 months to female transgenic and wild-type mice of 7 months of age (n ¼ 12/group). Activated microglia was measured by Iba1 immunohistochemistry, area fraction and cell number evaluation. In vitro experiments were carried out on microglia enriched rat primary cultures, activated by LPS 0.1mM for 8 h, and CHF5074 pretreatment was performed at 1, 3 and 10mM one hour before LPS administration. TNFalfa, IL-6, IL-12 and RANTES were measured by ELISA in the cell culture medium. Results: In vivo, quantification of immunoreactivity indicated that compared to transgenic controls, wild-type animals had much lower activated microglia (cell count and area fraction, P<0.05). Compared to Tg2576 control mice, activated microglia in the cerebral cortex was significantly (P < 0.05) reduced by CHF5074 treatment. In vitro experiments indicated that RANTES up-regulation by LPS was significantly attenuated by CHF5074 3 and 10 mM. Conclusions: This results of this study showed that CHF5074 significantly inhibits neuroinflammatory activity mediated by microglia. RANTES (regulated upon activation, normal T cell expressed and secreted) regulation could support a specific role in microglia-astrocyte cross-talk.

P2-116

ACTIVATION OF NLC4 INFLAMMASOME IN PRIMARY RAT ASTROCYTES BY PALMITATE ENHANCES ALZHEIMER’S DISEASE–LIKE CHANGES IN PRIMARY NEURONS

Li Liu, Christina Chan, Michigan State University, East Lansing, Michigan, United States. Background: Alzheimer’s disease is a devastating neurodegenerative disease and characterized by accumulation of A b, but the mechanisms leading to neuronal dysfunction has not been fully elucidated. Many studies suggest that inflammatory and immunological processes from astroyctes and microglia are central to the initiation and progression of Alzheimer’s disease, with interleukin 1 b (IL-1 b) as a key cytokine in the inflammatory response. IL-1 b maturation involves activation of a protein complex, inflammasome. Activation of different receptors of inflammasome is cell specific, and dependent upon the type of stimulation. Our studies show that saturated free-fatty acid, palmitate (PA) increase IL-1 b secretion by astrocytes, which in turn, upregulates A b level in primary rat neurons. However, the molecular mechanism by which PA induces IL-1 b secretion by the astrocytes is unknown. Methods: Western blot, Quantitative real time polymerase chain reaction, enzyme-linked immunosorbent assay, immunocytochemistry and RNA interference were performed. Results: Inflammasome includes caspase1, apoptosis-associated speck-like protein containing a CARD (ASC) and cytoplasmic receptors of NLR family. Our data demonstrate that PA induces IL-1 b production from primary rat astrocytes. The active form of caspase1 involved in the maturation of IL-1 b is upregulated upon PA treatment, and silencing caspase1 decreases IL-1 b level. ASC is also increased significantly and silencing ASC downregulates the IL-1 b level. This indicates that components of the inflammasome are involved in the maturation of IL-1 b in primary astrocytes. Further studies confirm that cytoplasmic receptors, namely NOD-like receptor family CARD doman containing 4 (NLRC4), is required for activation of the inflammasome and the release of IL-1 b by primary rat astrocytes upon PA treatment. Conclusions: Our findings indicate that the activation of the NLRC4 inflammasome in primary

rat astrocytes by PA may play a critical role in the inflammatory response in Alzheimer’s disease.

P2-118

LIVER X RECEPTORS LIGAND AND MINOCYCLINE: EFFECTIVENESS OF COMBINED DRUG THERAPY IN AN ALZHEIMER’S TRANSGENIC MOUSE MODEL

Valeria Flaque1, Claudio Cuello2, Martin Bruno3, 1Catholic University of Cuyo, San Juan, Argentina; 2McGill University, Montreal, Quebec, Canada; 3Catholic University of Cuyo, San Juan, Argentina. Background: Alzheimer’s disease (AD) is a multifactorial disorder (combination of genes and environmental factors) and apparently involves several different etiopathogenic mechanisms. Currently, there is no effective treatment to halt the progression or prevent the onset of AD. A number of theories involving various risk factors such as diet, lifestyle, socioeconomic status, genetic predisposition and head injury have been proposed, but the degree of contribution of each factor is still controversial. Along with age, several factors appear to be widely acknowledged as early risk factors for development of AD including chronic neuroinflammation, brain oxidative damage, hypercholesterolemia and Apolipoprotein E. On the other hand, there is a solid body of scientific evidence that the progressive brain accumulation of the peptide Abeta is key to the AD neuropathology. In recent studies, the initiation and progression of AD has been linked to neuroinflammation, cholesterol metabolism (including APOE activity) and Abeta accumulation, processes that can be modulated by minocycline and liver x receptors (LXRs). Methods: This study is aimed to test the effectiveness of the combined daily administration of minocycline with LXRs agonist (during four weeks) to establish the extent of the benefits of the combined therapy (synergism), as opposed to monotherapy with the two above-mentioned agents in our AD-like transgenic mouse model coded McGillThy1-APP. Results: We have been able to demonstrate and replicate the known beneficial CNS effects of minocycline treatment as an antioxidant and anti-inflammatory agent in our transgenic AD mouse model. Moreover, we have confirmed that Liver X receptor (LXRs) agonists’ treatment in our AD transgenic mouse model reverse cognitive deficits and facilitated the proteolytic degradation of Abeta, without inducing hepatic steatosis and hypertriglyceridemia. When combined, animals receiving both agents simultaneously, displayed a marked reduction of cortical peroxynitrite-mediated oxidative damage, lowered burden of Ab and decreased pro-inflammatory markers (IL-1b). Conclusions: This preliminary study provides evidence of the benefits of the combined therapy (synergism), as opposed to monotherapy with minocycline and LXRs agonist in our AD-like transgenic mouse model. These observations suggest that the combined administration may represent a promising therapeutic opportunity.

P2-119

MYD88 IS DISPENSABLE FOR CEREBRAL AMYLOID PATHOLOGY AND NEUROINFLAMMATION IN PSAPP TRANSGENIC MICE

Tara Weitz1, Melanie Das1, Kavon Rezai-Zadeh1, David Gate1, Terrence Town2, 1Cedars-Sinai Medical Center, Los Angeles, California, United States; 2Cedars-Sinai Medical Center/UCLA, Los Angeles, California, United States. Background: Activated microglia are associated with amyloid plaques in transgenic mouse models of cerebral amyloidosis and in human Alzheimer disease (AD); yet, their exact role in AD pathogenesis remains unclear. It has been suggested that microglia play dual roles depending on the context of activation - contributing negatively to disease pathogenesis by secreting pro-inflammatory innate cytokines or performing a beneficial role via phagocytosis of extracellular Ab. An important question remains as to how and when microglia recognize and respond to Ab peptides. In this regard, toll-like receptors, the majority of which signal through the adaptor protein myeloid

Poster Presentations: P2 differentiation factor 88 (MyD88), have been suggested as candidate Ab innate immune recognition receptors. It was recently reported that deficiency of MyD88 in the doubly transgenic PSAPP mouse model of cerebral amyloidosis led to reduced cerebral amyloid pathology and microglial activation. Methods: In order to assess a putative role of MyD88 in cerebral amyloidosis and glial activation in PSAPP mice, we crossed MyD88 deficient (MyD88 -/-) mice with PSAPP mice (both on a pure C57BL/6 background), interbred F1 offspring, and analyzed PSAPP-MyD88 +/+, PSAPP-MyD88 +/- and PSAPP-MyD88 -/- cohorts. Mice were aged to w13 months and analyzed for AD-like pathology and glial cell activation. Results: Biochemical analysis of detergent-soluble and -insoluble Ab 1-40 or Ab 1-42 in brain homogenates did not reveal significant between-groups differences. Further, no significant differences were observed on 1) amyloid plaque load by 4G8 immunohistochemistry, and 2) soluble fibrillar Ab by conformation-specific OC antibody. Additionally, neither activated microglia nor astrocytes differed between the three groups. Conclusions: Our data suggest that MyD88 signaling is not integrally involved in Ab-induced microglial activation in PSAPP mice and that signaling through MyD88 does not significantly impact the nature or extent of b-amyloidosis in PSAPP mouse brains.

P2-120

INFLAMMASOME-MEDIATED ACTIVATION OF CASPASE-1 AND CASPASE-6 IN PRIMARY HUMAN NEURONS

Vikas Kaushal, Andrea LeBlanc, McGill University, Montreal, Quebec, Canada. Background: Caspase-6, a key cysteine protease, is up-regulated in Alzheimer disease (AD) neurons and causes axonal degeneration in the absence of cell death. The activation of Caspase-6 is regulated upstream by the inflammatory Caspase-1, a component of a multiprotein complex known as inflammasomes; consisting principally of a nucleotide-binding oligomerization domain (NOD)-like receptor, apoptosis-associated speck-like protein containing caspase recruitment domain (ASC) and Caspase-1. While inflammasome function and intrinsic innate immunity have been well investigated in macrophages, little is known in neurons. In this study we examine the role of intrinsic neuronal immunity in regulating Caspase-6 activation. Methods: mRNA and protein expression of the main protein components of the inflammasomes were assessed by quantitative RT-PCR and western blots of primary human neurons. Purinergic P2X7 receptors (P2X7R) were targeted with the agonist benzylated ATP (BzATP) to induce the inflammasome. P2X7R-mediated pore formation was assessed by the uptake of the YO-PRO-1 dye. Caspase-1 and Caspase-6 activity was measured using the fluorogenic substrates ZYVAD-AFC and Z-VEID-AFC, respectively. Results: P2X7R mRNA was expressed in neurons but not in astrocytes. Treatment of neurons with BzATP induced a considerable uptake of the YO-PRO-1 dye and this uptake was completely inhibited by Brilliant Blue G, a specific P2X7R antagonist. These results indicate that P2X7R activation is opening the pannexin pore. Activation of the inflammasome by BzATP was confirmed by increased Caspase-1 activity. Quantitative RT-PCR showed that inflammasome components, IPAF-1, AIM-2, NALP1, and ASC mRNA were expressed in neurons but only the NALP1 mRNA increased with BzATP treatment. No NALP3 or NALP6 inflammasome receptor expression was observed. BzATP-induced a sequential increase of Caspase-1 YVADase activity and Caspase-6 VEIDase activity within 30 min and 1 hr of treatment, respectively. Blocking Caspase-1 with inhibitor Z-YVADfmk reduced Caspase-6 activity in BzATP-treated neurons, confirming that inflammasome formation and Caspase-1 activity are upstream of Caspase-6 activation. Conclusions: These results provide the first characterization of inflammasome components in neurons. Their presence implies that neurons have intrinsic innate immune mechanisms involved in Caspase-1 and Caspase-6 activation. Furthermore, these mechanisms could explain the Alzheimer brain associated increased levels of interleukin-1-beta, a cytokine produced only by Caspase-1.

P2-121

P303 CHROMOSOME 21-ENCODED MIRNA-155 UPREGULATION AND COMPLEMENT FACTOR H (CFH) DEFICITS IN DOWN SYNDROME (DS) AND ALZHEIMER’S DISEASE (AD)

Y.Y. Li1, S Bhattacharjee1, J.M. Hill1, P.N. Alexandrov2, A.I. Pogue3, Walter Lukiw4, 1Louisiana State University, New Orleans, Louisiana, United States; 2Russian Academy of Medical Sciences, Moscow, Russia; 3 Alchem Biotech, Toronto, Ontario, Canada; 4Louisiana State University Neuroscience Center, New Orleans, Louisiana, United States. Background: Down syndrome (DS), a congenital disorder associated with cognitive impairment and early-onset Alzheimer’s disease (AD), is a progressive genetic pathology resulting from full or partial triplication of chromosome 21. Both DS and AD brain is typified by activated microglia, increases in inflammatory signaling, and pathogenic alterations in the expression of glycoproteins that regulate the innate immune system. Methods: Bioinformatics; DNA array and micro RNA (miRNA) array profiling; Northern dot blot analysis; post-mortem tissue analysis; primary human brain cell culture; qRT-PCR. Results: In these studies, a screening of micro-RNA (miRNA) from AD and DS brain and peripheral tissues indicated a significant up-regulation of a chromosome 21-encoded miRNA-155, and a decrease in the abundance of the miRNA-155 mRNA target complement factor H (CFH), an abundant regulatory glycoprotein and a repressor of the innate immune response. Stressed primary human neuronal - glial cells indicated both miRNA-155 increase and CFH down-regulation, an effect that was reversed using anti-miRNA-155 (AM-155). Ancillary regulation of CFH expression was found to be moderated also by miRNA-125b and miRNA-146a, depending on brain cell type. Conclusions: These results are the first to show regulation of a human innate immune system regulator, CFH, by multiple miRNAs in human brain cells and tissues. These findings further suggest that immunopathological deficits associated with AD and DS can, in part, be explained by a miRNA-125b, miRNA-146a and/or miRNA-155-mediated down-regulation of CFH that appears to contribute to both the brain and systemic immune pathology.

P2-122

THE ROLE OF TLR-4 MODULATE MICROGLIA ACTIVATION REDUCES NEURONAL PLASTICITY AND COGNITIVE FUNCTIONS IN ALZHEIMER’S DISEASE

Yi-Ling Liu, National Yang-Ming University, Taipei, Taiwan. Background: The inflammatory reaction includes a string of complex biological response in Alzheimer’s disease (AD) pathogenesis, which may contribute to inflammation, neurodegeneration, and cognitive decline to the brain. Microglia regulate the main immune response in the brain, and it’s ratio of M1/M2 can affect the neurotoxic and neuroprotective processing. Toll-like receptors (TLRs) mediate immune response and are critical in amyloidogenic diseases. TLR2, TLR4, TLR8, and TLR9 had been demonstrated to participate in the amyloid-pathology. Here we hypothesize that the accumulation of amyloid-beta (Ab) might activate M1 microglia through TLRs and result in cognitive disorder. Methods: To evaluate TLR-mediated microglia activation process in neuronal death, we use mix culture system and brain slices from APP transgenic mice to calculate the level of activated M1/M2 microglia by immunocytochemistry, flow cytometry, immunohistochemistry (IHC), tomato lectin histology and eosin stain. To identify Aß-induced molecular changes, we use ELISA, immunoblot and IHC to measure Ab level, amyloid plaque deposition and markers for synaptic activity and inflammation. To demonstrate functional changes in transgenic mice, we evaluate the behavior and neuroplasticity deficits by Morris water maze and long-term potentiation. Results: Ab increased neuroinflammation by activate microglia in vitro and in vivo. The water maze test demonstrated that the transgenic mice have impaired learning and memory. Conclusions: The microglia activation through TLR signaling pathway(s) may be a potential therapeutic target for the clearance ability of Ab-deposits in the brain.