P2-461: Selective modulation of gamma-secretase cleavage of the amyloid precursor family of proteins

Poster Presentations P2: Background: In the present study, the effects of sub-chronic rolipram treatment in an object recognition task in 3-month-old male rats were investigated. Rats remember which object they have explored in a previous trial (T1) when they are tested one hour later (T2). However, when tested twenty-four hours later, they do not remember which object was presented to them in the first trial. Drug treatments may improve discrimination performance after 24 h, i.e. improve memory for the familiar object. Methods: Rats were sub-chronically treated with 0.5 mg/kg rolipram (p.o.) once daily for 5 consecutive days and tested with a 24 h delay between T1 and T2. Memory performance in the object recognition task was assessed before, during and after sub-chronic treatment. In addition, we investigated whether the timing of the final dose, i.e. 24 h, 1 h or 6 h before T1, had an effect on memory performance. Results: During sub-chronic treatment, i.e. after 2-3 days of rolipram treatment, moderate effects on memory performance were observed. After 5 days of teatment, rolipram improved longterm memory performance regardless of when the final injection was given. Determination of plasma and brain levels of rolipram at T1 revealed no detectable levels, when the compound was given 24h before T1. Conclusions: Thus, the effect of rolipram on memory acquisition in this task outlasts any acute effects of the compound which would depend on its plasma and brain levels. These long-lasting memory enhancing effects of rolipram might be explained by long-lasting neuronal changes by subchronic treatment due to recurring activation of the cAMP/PKA/CREB pathway leading to CREB phosphorylation, and therefore, to an persistent strengthening of synaptic plasticity. P2-460

CHRONIC LITHIUM CHLORIDE TREATMENT REDUCES A␤ LOAD AND AMELIORATES LEARNING AND MEMORY DEFICITS IN A MOUSE MODEL OF FAMILIAL ALZHEIMER’S DISEASE

Maria Cristina Rosi, Anna Fiorentini, Cristina Grossi, Ilaria Luccarini, Fiorella Casamenti, University, Florence, Italy. Contact e-mail: [email protected] Background: Glycogen synthase kinase 3␤ (GSK-3␤) is a major kinase implicated in the pathogenesis of Alzheimer’s disease (AD) and reducing its activity may have therapeutic efficacy. Lithium is an effective, albeit weak, inhibitor of GSK-3 and a commonly used drug to treat bipolar disorders. Methods: Here, the effects of chronic LiCl administration were investigated in TgCRND8 mice, carrying combined amyloid precursor protein (APP) Swedish (K670M/N671L) and Indiana (V717F) mutations and exhibiting cerebral amyloid deposition and cognitive deficits since 3 months of age. TgCRND8 (tg) mice (n⫽16) at the early/pre-symptomatic age of 2 months and age-matched wild-type (wt) mice (n⫽16) received i.p. injections of either 0.6 M LiCl (10 ␮l/g of body weight) or sterile NaCl (10 ␮l/g of body weight) daily for 5 weeks. Results: This treatment paradigm was well tolerated and no adverse effects, including changes in the weight of the mice, were observed. Similar steady-state levels of total GSK-3␤ were found in treated and untreated mice. In contrast, lithium administration increased inactive phospho-GSK-3␤Ser9 levels and decreased active phosphoGSK-3␣Tyr279 and phospho-GSK-3␤Tyr216 expression in tg mice, indicating inhibition of GSK-3 activity. The immunohistochemical detection and quantification of A␤(1-42) deposits demonstrated a significant reduction of amyloid plaque burden both in the motor cortex (-33,3% for plaque number, p⬍0.05, and - 48% for maximum plaque area, p⬍0.02; unpaired Student’s t-test) and in the hippocampus (-56% for maximum plaque area, p⬍0.001; unpaired Student’s t-test) of LiCltreated TgCRND8 mice, with respect to age-matched vehicle-treated tg mice. In the hippocampus of LiCl-treated tg mice a reduction of GFAPimmunoreactive cells in the A␤ plaque surroundings was found as well. Interestingly, LiCl treatment significantly improved working memory performances in the “step down” inhibitory avoidance test (p⬍0.001, two-way-ANOVA ⫹ Bonferroni’s post-test) and ameliorated visual and spatial learning abilities in the Morris water maze task (p⬍0.01, one-

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way-ANOVA ⫹ Bonferroni’s post-test). Conclusions: Overall, these data point to lithium or GSK-3 inhibitors as effective therapeutic tools in ameliorating AD pathology. It remains to be determined whether lithium treatment will be able to affect A␤ and/or tau pathology as well as memory functions in later stages of disease progression. P2-461

SELECTIVE MODULATION OF GAMMASECRETASE CLEAVAGE OF THE AMYLOID PRECURSOR FAMILY OF PROTEINS

Carlo Sala Frigerio1, Thomas L. Kukar2, Pamela Osenkowski3, Paul C. Engel1, Michael S. Wolfe3, Todd E. Golde2, Dominic M. Walsh1, 1University College Dublin, Dublin, Ireland; 2 Mayo Clinic College of Medicine, Jacksonville, FL, USA; 3Harvard Medical School and Brigham and Women’s Hospital, Boston, MA, USA. Contact e-mail: [email protected] Background: Therapeutic inhibition of gamma-secretase should prove useful for the treatment of Alzheimer’s disease, however developing effective inhibitors presents a serious challenge, since gamma-secretase is known to process at least 33 other substrates, 2 of which are the APP homologs, amyloid precursor like protein 1 (APLP1) and 2 (APLP2). Genetic ablation studies have revealed that knock out of APLP2 in combination with ablation of either APLP1 or APP results in perinatal lethality, whereas APLP1(-/-)/APP(-/-) mice are apparently normal. These findings suggest that APP and APLP2 may have distinct physiological functions and that APLP2 has the key physiological role among the family members. Consequently, useful gamma-secretase inhibitors should specifically target APP processing while sparing cleavage of APLPs. Methods: To identify inhibitors capable of selectively modulating gamma-secretase cleavage of APP, we have optimized an in vitro microsomal assay to monitor generation of APP and APLP intra-cellular domains (ICDs). Results: Time course analysis of ICD production revealed that APLP1 (t1⁄2max ⫽ 3 min, where t1⁄2max ⫽ the time required to achieve half the maximum level of ICD production) is processed faster than APLP2 (t1⁄2max ⫽ 9 min) and APP (t1⁄2max ⫽ 22 min). Using the t1⁄2max time points we tested the effect of structurally diverse molecules on ICD production. These included active-site directed inhibitors and non-steroidal anti-inflammatory drugs. As expected, active site-directed inhibitors similarly inhibited processing of all 3 proteins, whereas fenofibrate, a compound known to increase the Abeta42/Abeta38 ratio, had no effect on ICD production. Surprisingly, a related molecule, 2-[3-(3,5-dichlorophenoxy)phenyl]propanoic acid (referred to as FT-9), differentially inhibited the processing of APP and APLP2 compared with APLP1. For instance, 500 micromolar FT9 caused a 75% reduction in gamma-secretase cleavage of both APP and APLP2, but only a 25% reduction in APLP1 ICD production. Conclusions: Identification of a compound that inhibits gamma-secretase processing of APP but which has little effect on APLP1, coupled with our finding that the kinetics of gamma-secretase cleavage are different for the members of the APP family, suggests that it may be possible to develop specific inhibitors of APP gamma-cleavage while sparing the processing of other substrates. P2-462

SEN12333 (WAY-317538), A NOVEL ALPHA7 NICOTINIC RECEPTOR AGONIST WITH NEUROPROTECTIVE EFFICACY: IMPLICATION FOR NEURODEGENERATIVE DISEASES THERAPY

Carla Scali1, Marco Gianfriddo1, Renza Roncarati1, John Dunlop2, Georg C. Terstappen1, 1Siena Biotech S.P.A., Siena, Italy; 2Wyeth Research, Princeton, NY, USA. Contact e-mail: [email protected] Background: There is a large body of literature demonstrating nicotine-mediated neuroprotection in both in vitro and in vivo models. More recently a role for nicotinic acetylcholine receptors of the alpha7 type have been implicated in these neuroprotective effects although the underlying mechanisms remains to be elucidated. Methods: Using a functional FLIPR-based calcium assay employing the rat alpha7