CHRYSINA: FLAVONOID FOR THE MANAGEMENT OF MEMORY IMPAIRMENT IN MICE

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Poster Presentations: Tuesday, July 26, 2016

(IFNg, IL1b, IL6, TNFa, TGFb1, IL10, IL4R, COX2, NOS2, ARG1, CD206, CD86, TREM2, CD163, CHI3L1) and pre- (synaptophysin) and post-synaptic (PSD95) markers were measured by RT-PCR and ELISA, respectively. Results: In AD, relative to controls, there was a significant increase in levels of Ab (p<0.001), pTau (p<0.001), CHI3L1 (p¼0.015) and microglial markers related to phagocytosis (CD68, p¼0.037), antigen presentation (HLA-DR, p¼0.044) and immune response (FcgRI, p¼0.002). We also observed a significant association between FcgRIIa and FcgRIII and CHI3L1 and FcgRIII in AD (AD- and AD+); and between FcgRI and FcgRIII in ASI cohorts (Ctrl+ and AD+). We showed that ASI significantly decreases the expression of FcgRIII (p¼0.03) and elevates the expression of anti-inflammatory genes IL4R (p¼0.04) and CHI3L1 (p¼0.012) in AD cases (AD+ vs. AD-). ASI did not affect Ab, pTau or synaptic proteins. Conclusions: Our findings confirm our previous observations as well as genetic studies that indicate a role for microglia in AD. Interestingly, ASI appears to promote immunosuppression without affecting AD pathology or synaptic proteins. This may be driven at least in part through communication between microglial cells via the FcgR, key receptors involved in the peripheral immune response. P3-107

ALTERATIONS IN DNA METHYLATION CONTRIBUTE TO NEUROINFLAMMATION IN ALZHEIMER’S DISEASE

Roxana Wiswell1, Rafael Guerrero-Preston2, Peter Cronin1, Jennifer Mott1, Paula Desplats1, 1University California San Diego, La Jolla, CA, USA; 2 Johns Hopkins University, Baltimore, MD, USA. Contact e-mail: [email protected] Background: Inflammation is a major component of Alzheimer’s disease (AD) pathology, implicated in amyloid deposition and associated with dementia and cognitive impairment. Emerging data show that altered epigenetic mechanisms also contribute to neurodegeneration. AD postmortem brains have acceleration of agerelated methylation changes and genes associated with increased disease susceptibility are differentially methylated in AD, even at early stages. Although epigenetic factors have been implicated in the production of pro-inflammatory cytokines, the role of DNA methylation on neuroinflammation is yet poorly understood. The goal of this study was to investigate the methylation status of inflammation-related genes in AD brains, progressing from mild cognitive impairment (MCI) to dementia. Methods: Genome-wide DNA methylation was profiled on postmortem frontal-cortex samples from MCI and AD cases and age and sex-matched control subjects (n¼24 cases /group) using Human Methylation450k microarray. Analysis of differential methylation in MCI and AD brains was performed in minfi using bump-humpting and block finder functions. Changes in methylation at individual CpGs were determined with Partek Genomics Suite computing for clinical diagnosis, gender and age in multivariate ANOVA analysis. Direct effects of DNA methylation changes on transcription of inflammatory genes were tested on human primary microglial cultures. Results: DNA methylation alterations were more prominent in MCI, with 35 differentially methylated blocks (DMBs) identified at q<0.05, in contrast to 15 DMBs in AD brains. GSEA analysis of DMBs showed significant enrichment for the common regulatory factors TCF3/LEF1, which mediates signals from TNFa; NRF1, implicated in insulin resistance, diabetes and inflammatory pathways; and NFAT, that mediates inflammation and calcium imbalance and selectively affected in AD brains. In addition, individual CpGs at IL13; IL17RB; IL3; IKBKAP; TNFRSF8; CXCL17 and IKBKG showed altered methyl-

ation in MCI and AD. Pharmacological modulation of DNA methylation in human primary microglial cultures elicited transcriptional changes of TFN-associated inflammation genes, including some that showed differential methylation in AD brains. Conclusions: Aberrant DNA methylation contributes to AD pathology by modulating the expression of inflammatory genes. Regulation of neuroinflammatory pathways involves the coordinated action of methylation and specific transcription factors. These alterations appear at early disease stages and may have a role on progression. P3-108

BENEFICIAL EFFECTS OF ANTIINFLAMMATORY, RNS60, IN AGED APPSWE/ PS1DE9 MICE

Qiaoqiao Shi1, Bin Liu1, Angela Sze Man Hung2, Kevin X. Le1, Barbara Caldarone1, Supurna Ghosh3, Richard Watson3, Cynthia A. Lemere1, 1Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA; 2Institute of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong, China; 3Revalesio Corporation, Tacoma, WA, USA. Contact e-mail: [email protected] Background: RNS60 (Revalesio Corp., Tacoma, WA), is electroki-

netically processed saline containing charge-stabilized nanostructures (oxygen-filled nanobubble core surrounded by layers of positive and negative electrical charges). RNS60 has been shown to have anti-inflammatory effects and protect against neurodegeneration and memory decline in the Alzheimer’s disease (AD) 5XFAD mouse model (Roy et al., 2014; Modi et al., 2014). Here, we examined the effects of RNS60 on behavior and AD pathogenesis in aged AD-like transgenic (Tg) mice. Methods: Aged, w16 month-old APPswe/PS1dE9 Tg mice were treated with 300 ml intraperitoneal (i.p.) RNS60 (n¼13) or Normal Saline (NS; n¼8) 3 days per week for 10 weeks. Wildtype (WT) mice were treated with i.p. NS (n¼8). Results: We found 1) Tg mice were significantly impaired in Contextual Fear Conditioning (CFC) training and Water T Maze (WTM) compared to age-matched WT mice at 18.5 months of age. RNS60 treatment improved freezing time during CFC training (p¼0.059) compared to NS-treated Tg mice but had no effect on acquisition or reversal learning in the WTM. 2) RNS60treated Tg mice had significantly less hippocampal Ab42 deposition compared to NS Tg controls; however, Ab levels in whole brain homogenates were not significantly different between the Tg groups. 3) Pre- vs. post-treatment 18F-GE180 TSPO PET imaging revealed that RNS60 reduced glial activation. Microglia/macrophage immunolabeling in brain was significantly reduced in RNS60-treated Tg mice compared to NS Tg controls, while astrocyte staining was non-significantly reduced. 4) Anti-inflammatory cytokine IL-10 was elevated in brain in RNS60-treated Tg mice while pro-inflammatory cytokine TNF-a was reduced. 5) Synaptic puncta density and synaptic protein levels were partially but significantly rescued in RNS60-treated Tg mice compared to NS Tg controls. 6) Mature-BDNF and downstream p-CREB and p-ERK were partially normalized to WT levels in RNS60-treated Tg mice. 7) Phospho-GSK3b was increased in RNS60-treated Tg mice, indicating reduced GSK3b activity. Conclusions: RNS60 treatment reduced inflammation and AD pathology, and modestly improved cognition, in aged AD mice. Lack of effect on the WTM may be explained by the advanced age (w18.5 mo) of the mice at the time of testing. P3-109

CHRYSINA: FLAVONOID FOR THE MANAGEMENT OF MEMORY IMPAIRMENT IN MICE

Pawan Krishan1, Nitin Bansal2, Harpreet Kaur3, 1Punjabi University, Patiala, India; 2ASBASJSM College of Pharmacy, Bela, Ropar, India;

Poster Presentations: Tuesday, July 26, 2016 3 ASBASJSM College of Pharmacy, Bela, Ropar, India. Contact e-mail: [email protected]

Background: Chrysin is a naturally occurring flavonoid may be ex-

tracted from honey and propolis. Chrysin has been reported to possess multiple biological activities including anti-inflammatory and antioxidant properties. The present study aimed to explore the role of chrysin in the management of memory deficit in mice. Methods: Swiss albino mice of either sex aged 6-8 weeks and weight 25-30 g were used. The experimental protocol was duly approved by the institutional animal ethics committee. Chrysin was administered in two doses (30 and 60 mg/kg; po) to mice for 28 successive days. Memory impairment was induced by two methods: (i) Lipopolysaccharide (LPS; 1 mg/kg; i.p.) induced amnesia; (ii) alprazolam (Alp; 0.5 mg/kg; i.p.) induced amnesia. Elevated plus maze and morris water maze tests were used to evaluate memory. After behavioural evaluation, the animals were sacrificed to estimate brain TBARS, AChE activity, GSH, Nitrite, SOD activity and catalase activity. Results: Administration of chrysin for 28 days daily prevented the LPS and Alp induced enhancement of transfer latency of mice in elevated plus maze test and reduced the escape latency on 4th day and increased the time spent in target quadrant on 5th day of morris water maze test as compared to control group animals. Furthermore, decrease in brain TBARS, nitrite levels as well as AChE activity and increase in GSH, SOD and catalase activity in chrysin treated group. Conclusions: Chrysin may prove to be useful remedy for the management of memory owing to its possible neuroprotective and antioxidant properties. P3-110

DECIPHERING THE ROLE OF NEUROINFLAMMATION IN THE EARLY EVENTS OF ALZHEIMER’S DISEASE

Joshua Keene, Britanny Alexis Granett, Emily Michelle Crombez, Yannick Marchalant, Central Michigan University, Mount Pleasant, MI, USA. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is a slowly debilitating neurodegenerative disease where different factors can contribute to pathology progression. Among those, chronic inflammation could be considered, along with alterations in amyloid metabolism and tau protein hyperphosphorylation, as a major factor influencing synaptic function and neurodegeneration. Despite numerous studies have validated inflammatory mediators or the brain’s immune system in AD, the precise role of inflammatory processes in the disease pathophysiology is still highly controversial. Methods: 16 groups of animals (5xFAD mice, males and females) are subjected to foetal chronic inflammation using lipopolysaccharide (LPS) and/or early exposure (2-months old) to chronic inflammation using LPS in order to assess their effects on behaviour, hallmarks of AD’s progression and changes in neural cell function. Awide range of technical approaches aer used including behavioural tests, microsurgery, biochemistry, immunohistochemistry and molecular biology. Results: Plaques load, glial cells, neurogenesis and cell death are quantified using unbiased stereology to assess the role of inflammation on AD’s onset/ progression. ELISA are used to determine cytokines levels in the hippocampus and frontal cortex and western blots to determine the amyloid b species (and changes) in the different groups observed. Conclusions: this work aims at clarifying the role of neuroinflammation and its mechanisms in the very early phases of AD via a comprehensive approach that takes into account, using in vivo and in vitro experimental models, the timely interactions between immune/inflammatory processes and AD’s pathophysiological hallmarks. As no cure is currently available for AD, identifying and understanding

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major risk factors of the development of the disease is crucial. This approach can open in the short-term new avenues for preventive/delaying strategies in order to decrease the prevalence of the disease in the next decades. P3-111

DO POSTANESTHETIC MEMORY DEFICITS AND ALZHEIMER’S DISEASE SHARE A COMMON SIGNALING PATHWAY?

Kirusanthy Kaneshwaran1, Sean Haffey1, Gang Lei1, Dian-Shi Wang1, Beverley A. Orser1,2, 1University of Toronto, Toronto, ON, Canada; 2 Sunnybrook Health Sciences Centre, Toronto, ON, Canada. Contact e-mail: [email protected] Background: A tonic inhibitory current generated by extrasynaptic

GABAA receptors (GABAAR) in the hippocampus inhibits learning and memory processes (Proc Natl Acad Sci, 2004; J Neurosci 2010). An increase in the tonic inhibitory current may mediate memory deficits following anesthesia (Journal Clin Invest, 2014) and in Alzheimer’s disease (AD) (Nature, 2014). Inflammatory processes, including the activation of astrocytes, may increase the tonic current and contribute to these memory disorders (Journal Clin Invest, 2014; Brain, 2016). Postanesthetic memory loss and AD may share similar underlying pathways, as the pro-inflammatory cytokine, IL-1b, acutely increases the tonic current via a p38MAPK-dependent signaling pathway (Cell Reports, 2012) and levels of IL-1b are increased in the brains of AD patients (Curr Pharm Des, 2016). The goal of this study was to determine whether an IL-1b- and p38-MAPK-dependent inflammatory cascade mediates the increase in tonic GABA current. We also studied the role of the anti-inflammatory drug minocycline in preventing the anesthetic-induced increase in tonic current. Methods: Co-cultures of murine hippocampal neurons and cortical astrocytes were treated with drugs for 1 h then washed. Whole-cell voltage clamp techniques were used to record tonic GABA current 24 h after treatment. In other studies, mice were treated with etomidate (8mg/ kg, intraperitoneal) and brain samples were collected 24 h later. IL-1b levels were measured in the hippocampus using Western blotting. Results: Etomidate-induced increase in tonic GABA current was reversed by co-treatment with an IL-1b receptor antagonist, IL-1Ra (100 ng/mL), and a p38MAPK inhibitor, SB 203,580 (20 mM). IL-1b (60 ng/mL) and lipopolysaccharide (LPS, 100ng/ mL) persistently increased tonic GABA current. Minocycline (100 mM) prevented the etomidate-induced increase in tonic GABA current. Western blot showed that etomidate enhanced IL1b levels in hippocampal tissue. Conclusions: An inflammatory signaling pathway that involves IL-1b and p38-MAPK mediates etomidate-induced persistent increase in GABAAR-mediated tonic current that underlies memory deficits. Minocycline attenuates the increase in tonic GABA current. These results suggest that memory deficits after anesthesia and AD may, in part, share similar underlying mechanisms. These findings pave the way for better management and treatment of both pathologies. P3-112

HIGHER RATE OF ALZHEIMER’S DISEASE AMONG ARCTIC POPULATIONS IS DUE TO ACTIVATION OF BRAIN INFLAMMATORY PATHWAYS BY ELEVATED DIOXIN BODY BURDEN: MECHANISTIC APPROACH TO THE PROBLEM

Ilya B. Tsyrlov1, Sergey G. Krivoschekov2, 1XENOTOX, Inc., Scarsdale, NY, USA; 2Institute of Physiology, Russian Academy of Sciences, Novosibirsk, Russian Federation. Contact e-mail: [email protected]