P1-449: Aminopeptidase A is involved in amyloid beta peptide N-terminal truncation

Poster Presentations P1 contribute to cognitive and physiological deficits at an early stage of Alzheimer’s disease. P1-447

TIME-COURSE EFFECTS OF AMYLOID TOXICITY INDUCED BY A SINGLE ICV INJECTION OF A␤(25-35) ON THE GLUCOCORTICOIDS SYSTEM IN RATS

Anthony Brureau, Charleine Zussy, Brice Delair, Tangui Maurice, Laurent Givalois, Molecular Mechanisms in Neurodegenerative Dementias, U710 Inserm, University of Montpellier 2, EPHE, Montpellier, France. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is a neurodegenerative pathology characterized by the presence of senile plaques and neurofibrillary tangles, accompanied by synaptic and neuronal loss. The major component of senile plaques is a ␤-amyloid protein (A␤). Intracerebral injection of A␤ to rodents induces learning and memory impairments as well as neurodegeneration in brain area related to cognitive functions. In AD patients, it was also observed some endocrine modifications and more particularly changes in the hypothalamo-pituitary-adrenocortical (HPA) axis activity, which are characterized by an hyper-secretion of glucocorticoids. Methods: In this study, we assessed the time-course effects (during 6 weeks) of a single intracerebroventricular (icv) injection of aggregated A␤ fragment (25-35) at a dose of 10 ␮g/rat on the glucocorticoids system. The hippocampal, amygdala, frontal cortex and hypothalamic mRNA and protein levels (RT-PCR and western blot) of glucocorticoids receptors (MR and GR) were evaluated before and 1, 2, 3 and 6 weeks after the single icv injection of A␤(25-35). The effects of A␤ icv injection on the subcellular localization of glucocorticoids receptors were also evaluated by immunohistochemistry. Plasmatic concentrations of ACTH and corticosterone (CORT) were determined (RIA) before and after A␤(25-35) injection. The eventual modification of glucocorticoids feedback following the single icv injection of A␤(25-35) was estimated with the dexamethasone (DEX) suppression test. The icv injection of scrambled A␤(25-35) peptide was used as sham control. Results: The results show that aggregated A␤(25-35) affects differentially the expression of GR and MR over the time and the cerebral region considered. It seems that amyloid peptide induces a progressive localization of GR in the nucleolus over the time, which is more pronounced in the frontal cortex, the amygdala and the hippocampus than in the hypothalamus. Plasma concentrations of ACTH are significantly increased only 3 and 6 weeks after the single injection of A␤(25-35), while the plasma concentrations of CORT are significantly increased after 1, 2, 3 and 6 weeks. Conclusions: In conclusion, this study shows that A␤(25-35) seems to induce a glucocorticoids resistance that could initiate and/or contribute to cognitive and physiological deficits at an early stage of Alzheimer’s disease. P1-448

TIME-COURSE EFFECTS OF AMYLOID TOXICITY INDUCED BY A SINGLE ICV INJECTION OF A␤(25-35) ON BEHAVIOR, OXIDATIVE, ENDOPLASMIC RETICULUM AND MITOCHONDRIAL STRESSES AND ON CEREBRAL APOPTOTIC PROCESS IN RATS

Charleine Zussy, Anthony Brureau, Brice Delair, Tangui Maurice, Laurent Givalois, Molecular Mechanisms in Neurodegenerative Dementias, U710 Inserm, University of Montpellier 2, EPHE, Montpellier, France. Contact e-mail: [email protected] Background: Alzheimer’s disease is a neurodegenerative pathology characterized by the presence of senile plaques and neurofibrillary tangles, accompanied by synaptic and neuronal loss. The major component of senile plaques is a ␤-amyloid protein (A␤). Intracerebral injection of A␤ to rodents induces learning and memory impairments as well as neurodegeneration in brain area related to cognitive functions. Methods: In this study,

T351

we assessed the time-course effects (during 6 weeks) of a single intracerebroventricular (icv) injection of aggregated A␤ fragment (25-35) at a dose of 10 ␮g/rat, on body weight, general activity and body temperature rhythms, anhedonia and spatial short- and long-term memories (T-maze and Water maze tests). In the hippocampus, the frontal cortex, the amygdala and the hypothalamus, we analyzed oxidative stress (lipid peroxidation assay), endoplasmic reticulum and mitochondrial stresses and apoptotic process (caspases12, 9 and 3 variations, respectively) during the 6 weeks following the single icv injection of A␤(25-35). The icv injection of scrambled A␤(25-35) peptide was used as sham control. Results: The results show that aggregated A␤(25-35) failed to affect anhedonia, general activity and temperature rhythms over the time. By contrast, A␤(25-35) induces significant body weight decrease. The spatial short-term memory is significantly impaired 7, 14, 21 and 42 days after the 10 ␮g injection of the amyloid peptide. The spatial long-term memory is significantly changed 3 and 6 weeks after the amyloid peptide injection. A␤(25-35) differentially modifies over the time the oxidative, endoplasmic reticulum and mitochondrial stresses and apoptotic process in the hippocampus, the frontal cortex, the amygdala and the hypothalamus. Conclusions: In conclusion, this study confirms that A␤(25-35) induced memory impairment and shows that a single icv injection induces some central cellular modifications that could initiate and/or contribute to cognitive and physiological deficits at an early stage of Alzheimer’s disease. P1-449

AMINOPEPTIDASE A IS INVOLVED IN AMYLOID BETA PEPTIDE N-TERMINAL TRUNCATION

Jean Se´valle, Fre´de´ric Checler, IPMC CNRS, Valbonne, France. Contact e-mail: [email protected] Background: Amyloı¨d beta peptide (A␤) is the main component of the senile plaques, one of the main histopathological hallmarks of Alzheimer’s disease. A␤ is susceptible to N-terminal truncation by aminopeptidases. This cleavage is responsible for dramatical physical and chemical changes on A␤ structure and specially increase propensity to aggregate. Whereas full length A␤ toxicity remains highly discussed, several in vitro studies have shown that the N-term truncated forms of A␤ are cytotoxic. The aim of this study was to identify the aminopeptidase involved in A␤ truncation and investigate the impact of this truncation in cell death. Methods: For this study we used wild type, APPwt or APPswe over expressing HEK293 cells as well as wild type and APP -/fibroblasts. A␤ peptide immunoprecipitations were performed using a free N-terminal specific antibody. In order to specifically measure aminopeptidase A (APA) activity hydrolysis of a fluorimetric substrate in presence and absence of APA inhibitors was performed. The role of APA was determined by the measure of caspase-3 activity. Results: Our data clearly show that APA inhibition leads to increased full length A␤ immunoreactivity suggesting a protection of the N-terminus in HEK293 over expressing APPwt or APPswe. APA inhibitors reduce cell sensitivity to staurosporin induced apoptosis in HEK293 cells but not in APPwt ou APPswe overexpressing cells suggesting a protective effect of A␤ when it is present at physiological concentration. Furthermore, this phenotype is APP dependent since sensitivity to apoptosis is reduced after APA inhibitors treatment in wild type fibroblasts but not in APP -/- fibroblasts. Conclusions: Aminopeptidase A is involved in A␤ N-terminal truncation but might not be the only enzyme responsible for all N- truncations. Since the APA inhibitor used in his study is already available as a prodrug (design to target the renin-angiotensin system) which is orally biodisponible, this compound could be used in transgenic mice models of Alzheimer’s disease to investigate the benefits of such a treatment on the disease time course. P1-450

TOMOREGULIN-2 BINDS BETA-AMYLOID PRECURSOR PROTEIN AND INTRACELLULAR BETA-AMYLOID (A␤)

Lee-Way Jin, Hyun-seok Hong, UC Davis, Sacramento, CA, USA. Contact e-mail: [email protected]