GLYCOLYTIC CONTROL OF LXR-APOE REGULATORY PATHWAY IN ASTROCYTES

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Poster Presentations: P2

micro-aggregates and platelet-leukocyte interactions with WMH load suggest that thrombotic mechanisms in blood may contribute to development of ischemic white matter disease in postmenopausal women. P2-054

P2-056

DELETION OF 14-3-3GAMMA EXACERBATES DISEASE IN MOUSE MODELS OF NEURODEGENERATIVE PROTEINOPATHY 1

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Petra Steinacker , Stephen Meier , Evamaria G€orz , Alastair Aitken , Katrin Lindenberg1, Bernhard Landwehrmeyer1, Albert Ludolph3, Markus Otto1, 1University Ulm, Ulm, Germany; 2University of Edinburgh, Edinburgh, United Kingdom; 3Department of Neurology, Ulm University, Ulm, Germany. Contact e-mail: [email protected] Background: Neurodegenerative diseases have in common that specific proteins form aggregates in the affected tissues. Besides the specific proteins further proteins which often are associated with posttranslational protein quality control mechanisms are part of pathologic inclusions. 14-3-3 proteins, a family comprising 7 isoform that mainly bind to phosphorylated motifs of a large number of target proteins, regulate a wide variety of signaling cascades by acting as a chaperone, scaffold or adapter protein. There is evidence for a relation of 14-3-3 proteins and pathological aggregates, e.g. 14-3-3 colocalizes with neurofibrillary tangles in AD, with hyaline Lewy body-like inclusions in familiar ALS and is necessary for huntingtin aggregate formation. Aim of the present study is to determine if neurodegenerative disease course is modified when 14-3-3gamma is knocked out. Methods: We crossbred 14-3-3gamma deficient mice with mouse models of amyotrophic lateral sclerosis (SOD1(G93A) and Huntington’s disease (R6/2 mouse) and characterized disease course, survival, aggregate appearance and 14-3-3 isoform expression . Results: R6/2 mice deficient for 14-3-3gamma show an earlier weight loss and shorter survival time compared to R6/2 14-3-3WT littermates (p<0.0001). Aggregate appearance is not affected. SOD1(G93A) mice without 14-3-3gamma show exacerbation of the motor phenotype and earlier paresis, however the survival is not significantly different and the results are gender specific. Conclusions: In conclusion, 14-3-3gamma up-regulation could prolong life time in Huntington’s disease or improve motor performance in amyotrophic lateral sclerosis by mechanisms not known yet which seem not to be associated with the aggregate number or size. Further studies are needed to elucidate involved mechanisms and the reason for gender specific effects. P2-055

and the underlying mechanisms of these changes. Supported by a grant from the Health Research Council of New Zealand.

AGE-RELATED PLASMA L-ARGININE METABOLIC PROFILE CHANGES IN APP/PS1 MICE

Ping Liu, David Bergin, Yu Jing, Bruce Mockett, Wickliffe C. Abraham, Hu Zhang, University of Otago, Dunedin, New Zealand. Contact e-mail: [email protected] Background: L-arginine is a semi-essential amino acid that can be metabolized to form a number of bioactive molecules. Increasing evidence implicates altered L-arginine metabolism in the pathogenesis of Alzheimer’s disease. Methods: This study measured the plasma levels of L-arginine and its nine downstream metabolites (L-citrulline, L-ornithine, agmatine, spermidine, spermine, glutamine, glutamate and GABA) in 7 and 13 month old male APP SWE PS1 DE9 mice (APP/PS1) and their matched wild-type littermates (WT) using high-performance liquid chromatography and liquid chromatography mass spectroscopy. Results: We found that the plasma levels of L-arginine, L-citrulline, L-ornithine, glutamate and GABA increased with age in both the WT and APP/PS1 mice, but with no significant genotype differences. Furthermore, we observed increased agmatine levels with age, but no age-related changes in the polyamines putrescine, spermidine and spermine. There were increased agmatine levels and reduced putrescine and spermine levels in APP/PS1 mice at 7, but not 13, months of age when compared to their age-matched WT mice. No genotype or age effect was found for glutamine or spermidine. Conclusions: These results demonstrate altered plasma levels of L-arginine and its metabolites with age in a metabolite-specific manner, along with altered agmatine, putrescine and spermine levels in APP/PS1 mice mainly at 7 months of age. Future research is required to understand the functional significance

THE ROLE OF THE MARINE PLASMA IN THE ELDERLY WITH DEMENTIA AND HYPOVOLEMIA

Roberto Lacava1, Alberto Castagna2, Francesca Mazzei3, Maria Teresa Pontieri3, Antonino Maria Cotroneo4, Antonio Aversa5, Pietro Gareri6, 1ASP di Catanzaro, Tutela Anziani, Italy; 2AUSL Modena, Pavullo nel Frignano, Italy; 3RSA S. Francesco H. Settingiano, Catanzaro, Italy; 4ASL 2 Torino, Turin, Italy; 5Universita Sapienza, Roma, Italy; 6 Ambulatory Center for Dementia - ASP Catanzaro - Italy, Catanzaro, Italy. Contact e-mail: [email protected] Background: Our body consists primarily of water, present in all our tissues, its requirements vary with the type of physical activity, with age, the body and environmental temperature. In general, the elderly, along with a reduced dietary intake, it is verified that there is little support of liquids with a high risk of dehydration. Several studies have shown that in patients with dementia the risk of dehydration is higher and that the low intake of liquids, generally less than 1500 ml per day, is correlated with cognitive impairment and a reduction in the ability to perform activities of daily living. Therefore, it is clear that the need for the elderly to maintain a proper fluid and electrolyte balance in particular in those with dementia. The aim of our study was to evaluate the effects of rehydration in demented elderly hypovolemic patients through the use of " marine plasma " (sea water from the ocean micro-filtered at low temperature). Methods: A total of 40 patients were observed, all with dementia and severe cognitive A total of 40 patients were observed, all with dementia and severe cognitive impairment and hypovolemia in treatment with antipsychotics, who had poor oral fluid intake (<500 ml / day), average age 78 +6.4 years, MMSE 24.48 +8.54 +5.11 +1.81 NPI. 20 patients (MMSE 5:11 +2.08; NPI 24.83 +15) in a state of hypovolemia with the presence of confusion, delirium, agitation were hydrated orally with marine plasma (2 fld / day 6 times a week and constant oral hydration). After 30 days of treatment there was a reduction of symptoms (NPI 15 +5.49) and reduction of antipsychotic therapy. 20 patients (MMSE 5.28 +1.49, 24.89 +5.93 NPI in a state of hypovolemia with the presence of confusion, delirium, agitation were hydrated intravenously with physiological solution (500 ml / day 4 times / week and oral hydration) and hypovolemia in treatment with antipsychotics, who had poor oral fluid intake (<500 ml / day), average age 78 +6.4 years, MMSE 24.48 +8.54 +5.11 +1.81 NPI. Results: 20 patients (MMSE 5:11 +2.08; NPI 24.83 +15) in a state After 30 days of therapy and had a reduction in symptoms (NPI 14.94 +4.43) and continuity of antipsychotic therapy. In both groups it was noted a reduction of symptoms (measured with the NPI p < 0.000) , but it should be emphasized that where possible , oral hydration, as in our case, integrated marine plasma is definitely preferred. with the presence of confusion, delirium, agitation were hydrated orally with marine plasma ( 2 fld / day 6 times a week and constant oral hydration) . After 30 days of treatment there was a reduction of symptoms (NPI 15 +5.49) and reduction of antipsychotic therapy. 20 patients (MMSE 5.28 +1.49, 24.89 +5.93 NPI) in a state of hypovolemia with the presence of confusion, delirium, agitation were hydrated intravenously with physiological solution (500 ml / day 4 times / week and oral hydration) After 30 days of therapy and had a reduction in symptoms (NPI 14.94 +4.43) and continuity of antipsychotic therapy . In both groups it was noted a reduction of symptoms (measured with the NPI p < 0.000 ) , but it should be emphasized that where possible , oral hydration, as in our case, integrated marine plasma is definitely preferred. Conclusions: The isotonic sea water, having a composition qualitatively and quantitatively identical to the extracellular fluid may determine a favorable regulatory mechanism in exchange between the plasma, interstitial and intracellular sector participating in the restoration of the same cellular functionality. A consistent and appropriate use of marine plasma, in elderly patients, may, therefore, help restore proper homeostasis of the organism. P2-057

GLYCOLYTIC CONTROL OF LXR-APOE REGULATORY PATHWAY IN ASTROCYTES

Sachin P. Patil, Widener University, Chester, Pennsylvania, United States. Contact e-mail: [email protected]

Poster Presentations: P2 Background: AD is characterized by deleterious deposits of Ab and t proteins, which are central to AD pathogenesis. In addition to these pathophysiological changes, AD is also characterized by abnormal glucose metabolism that precedes neuropathological changes. Despite these data, the underlying molecular mechanism behind the potential cause and effect relationship between decreased glucose metabolism and increased Ab deposition is unknown. In this context, we hypothesized that decreased glucose metabolism may result in decreased Ab clearance and hence its increased deposition in AD brain. Here, it is noteworthy that Liver X receptor (LXR)-mediated Apolipoprotein E (APOE) lipidation and secretion plays a major role in cerebral Ab clearance. Therefore, we aimed to investigate glycolytic control of LXR-APOE pathway and identify its positive modulators for therapeutic purpose. Methods: The astrocytes expressing human APOE2/E3/E4 isoforms (from Dr. David Holtzman, Washington University) were used. The astrocytes were treated for 24 hrs with known LXR and RXR agonists (GW3965 and Bexarotene, respectively), either in presence or absence of inhibitors of glucose uptake and glycolysis (cytochalasin B and 2-deoxy-D-glucose, respectively). Furthermore, astrocytes were also treated with various FDA-approved drugs with potential to enhance glycolysis and in turn activating the LXR-APOE pathway. The uptake of glucose and the production of lactate were measured as markers of glycolysis. The level of secreted APOE was measured using human APOE ELISA, while lipidation status of APOE was measured using non-denaturing gel electrophoresis followed by western blotting. Results: The treatment of APOE3 astrocytes with Bexarotene increased APOE lipidation and secretion, which was inhibited by co-treatment with Cytochalasin B and 2-deoxy-D-glucose (Fig.1A). Furthermore, astrocytes were also treated with three drugs from three different categories viz., a b-blocker (Drug 1, 5mM), an anti-cancer agent (Drug 2, 5mM) and an anti-inflammatory agent (Drug 3, 2mM). The Drug 1 and 2 significantly enhanced glucose uptake and lactate production by astrocytes (Fig.1B). Interestingly, these two drugs also significantly enhanced APOE secretion (Fig.1B). The experiments with other drug molecules in APOE3 as well as APOE2 and E4 astrocytes are ongoing. Conclusions: Our data so far suggests potential role of glucose metabolism in APOE lipidation and secretion by astrocytes, further emphasizing our hypothesis.

Fig.1A. Co-treatment of astrocytes with Cytochalasin B (Cyto B) and 2-deoxy-D-glucose (2-DG) inhibits Bexarotene-induced APOE secretion and lipidation

Fig.1B. Drugs that increased astrocytic glucose uptake and lactate production also increased APOE secretion

P2-058

A GLYCOMICS APPROACH FOR ANALYZING CEREBROSPINAL FLUID

Stefan Gaunitz1, Sophia Schedin-Weiss2, Bengt Winblad2, Lars Tjernberg3, 1Karolinska Institute, Huddinge, Sweden; 2Karolinska

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Institute, Stockholm, Sweden; 3Karolinska Institute, Huddinge, Sweden. Contact e-mail: [email protected] Background: Glycosylation is the most common post-translational modification. Glycosylation modifications are more dynamic than protein levels and altered glycosylation pattern is often observed in disease. Traditionally, glycomics has been more laborious than proteomics due to extensive sample work up and manual interpretation of data. We hypothize that glycosylation may be altered in AD and that the proteins in the cerebrospinal fluid (CSF) should reflect such alterations. We have thus employed a simple protocol for glycan release from CSF and glycomic profiling with mass spectrometry. Methods: Cerebrospinal fluid samples were immobilized on polyvinylidene difluoride membranes and glycans were enzymatically relaesed from proteins with Peptide-N-Glycosidase F (PNGase F) enzyme. Alternatively glycans were released with PNGase F in solution and permethylated. Glycans were analyzed with nano electrospray ionization (ESI)-ion-trap mass spectrometry (ITMS) with automatic fragmentation of precursor ions. Different separation columns preceding detection and fragmentation in the ion trap were tested. Results: The preparation of glycans from CSF immobilized to polyvinylidene difluoride membranes proved to be simpler in comparison to protocols that release glycans in solution and involve permethylation derivatization. Porous graphitized carbon columns (PGC) allowed the separation of native isomeric glycan structures which was not possible for permethylated glycans separated on C18 columns. Conclusions: Glycomics on CSF samples immobilized to polyvinylidene difluoride membranes proved to be powerful and simple in comparison to protocols that release glycans in solution and involve permethylation derivatization. We suggest that glycomics on CSF may be used to study AD. It has not escaped our attention that altered glycosylation in CSF may be used as novel disease markers, increase the understanding of the disease and reveal novel therapeutic targets. P2-059

BACE1/2 INHIBITION DOES NOT REGULATE PANCREATIC BETA CELL FUNCTION AND MASS IN MICE

Peter O’Donnell1, Ulf Neumann2, Michael Pietropaolo1, Michael Fleming1, Xinyu Chen1, Xiurong Wang1, David Nettleton1, Gregory Bebernitz1, Vidya Kunjathoor1, 1Novartis, Cambridge, Massachusetts, United States; 2Novartis, Basel, Switzerland. Contact e-mail: [email protected] Background: Loss of pancreatic islet b-cell function and mass is a hallmark of type 2 diabetes. Inducing b-cell proliferation may restore function and mass in individuals with type 2 diabetes and ameliorate the disease. Akpinar et al (2005) demonstrated overexpression of full-length transmembrane protein 27 (TMEM27) induced b-cell proliferation in mice. TMEM27 is cleaved and inactivated by beta site amyloid precursor protein cleaving enzyme 2 (Bace2, Esterhazy, 2011). Furthermore Bace1/2 inhibition improved diabetes and islet b-cell function and mass in diabetic ob/ob mice. Correction of hyperglycemia was accompanied wtih increased circulating insulin levels, improved glucose tolerance and increased mass of b-cells. These effects were attributed to preventing cleavage of TMEM27 by Bace2 inhibition. We sought to confirm these effects using a potent in-house Bace1/2 inhibitor in promoting pancreatic b-cell function and mass in vitro and in vivo. Methods: We identified NB-360 as a very potent Bace 1/2 inhibitor (IC50s for Bace1 ¼ 3 nM, and Bace2 ¼ 2 nM). We evaluated NB-360 in vitro by measuring its effect on proliferation of b - cells using a murine pancreatic b-cell line, MIN6 cells, and primary dispersed rat islets containing b-cells. We also assessed the effects of NB-360 at 30, 60, 100 mg/kg, dosed orally twice a day for 28 days in diabetic ob/ob mice. We measured non-fasting glucose levels, glucose and insulinlevels during oral glucose tolerance tests and pancreatic insulin content as a surrogate for b-cell mass at the end of the chronic month-longstudy. Results: We report that NB360 did not block cleavage of TMEM27. NB-360 did not increase proliferation of either MIN6 cells or primary b-cells derived from rat islets in vitro. Consistent with these results, NB-360 did not improve hyperglycemia in diabetic ob/ob mice, or glucose tolerance or increase b-cell mass in the chronic study. Conclusions: Taken together, these data suggest that in contrast to previous publications,