P1-414: Regulation of amyloid-beta by cathepsin B and cystatin C

Poster Presentations P1 P1-414

REGULATION OF AMYLOID-BETA BY CATHEPSIN B AND CYSTATIN C

Li Gan, Binggui Sun, Yungui Zhou, Xin Wang, Seo-hyun Cho, Gladstone Institute of Neurological Disease, San Francisco, CA, USA. Contact e-mail: [email protected] Background: Accumulation of amyloid ␤ (A␤) peptides, especially A␤142, plays an important role in the pathogenesis of Alzheimer’s disease (AD). Strategies to lower A␤ levels include inhibiting its production or enhancing its degradation and clearance. We recently discovered that cysteine protease cathepsin B (CatB) reduces A␤ through C-terminal truncation of A␤1-42, suggesting that promoting endogenous CatB activity could reduce A␤ levels and have beneficial effects. The enzymatic activity of CatB is predominantly inhibited by its endogenous inhibitor cystatin C (CysC), whose polymorphism is associated with an increased risk of late-onset sporadic AD. Recent studies showed that human CysC inhibits A␤ fibril formation and lowers plaque load by directly binding to A␤. How CysC regulates levels of soluble A␤, however, remains unknown. The objective of the current study is to test the hypothesis that CysC may increase soluble A␤ levels by inhibiting CatB-mediated A␤-degradation. Methods: (1) We examined the effects of CysC reduction on levels of A␤, A␤1-42, premature mortality, and A␤-dependent neuronal deficits by crossing CysC knockout mice with transgenic mice overexpressing human amyloid precursor protein (hAPP) with familial AD-linked mutations; (2) We determined if CysC’s effects on soluble A␤ and associated neuronal deficits depend on CatB by examining the effects of CysC reduction in hAPP mice a CatB null background. Results: Genetic removal of CysC, which resulted in enhanced CatB enzymatic activities, significantly lowered soluble A␤ levels and relative abundance of A␤1-42 in both young and old hAPP mice. Remarkably, removing one allele of CST3 in hAPP mice was sufficient to lower soluble A␤, increase calbindin levels and the number of c-Fos-positive neurons in the dentate gyrus (DG), and prevent premature mortality. More over, the beneficial effects of CysC reduction depends on CatB since reducing CysC failed to lower the levels of soluble A␤ or to increase the DG calbindin expression in hAPP mice on a CatB null background. Conclusions: These findings demonstrate that the beneficial effects of CysC reduction are directly mediated by promoting CatB’s ability to truncate A␤, establishing CysC-CatB axis as a new pathway to regulate soluble A␤ and A␤-dependent neuronal deficits. Supported by NIA AG024447. P1-415

IDENTIFICATION OF A CANDIDATE GENE ASSOCIATED WITH A␤ TRANSPORT FROM BRAIN TO BLOOD

Jingwei Jiang, Masayasu Okochi, Shinji Tagami, Kouhei Nishitomi, Yufen Jin, Naohiro Itoh, Akio Fukumori, Taisuke Nakayama, Kohji Mori, Takashi Oguri, Kanta Yanagida, Masatoshi Takeda, Osaka University Graduate School of Medicine, Suita, Osaka, Japan. Contact e-mail: [email protected] Background: Local A␤ concentration in brain is thought to play an important role for inducing pathological roles of A␤. The local A␤ level was determined by balance of several factors, including the level of A␤ production, A␤ degradation and A␤ transport across brain-blood barrier (BBB). Although the mechanisms of A␤ generation and degradation have been well studied, mechanisms how A␤ is excreted across the BBB are still controversial. Here, we found a gene affecting the A␤ excretion across the BBB. Methods: To study the A␤ transport process, human A␤40/42 was injected into mice (brain lateral ventricle or blood vessels). Following chase-periods, we measured plasma and brain A␤ levels by ELISA. Results: We unexpectedly found a gene facilitating A␤ metabolism by accelerating A␤ excretion via BBB. Upon injecting A␤40/42 into wildtype mouse brains, A␤40/42 efflux to blood was reached at maximum after 30 minutes. While using the mutant mice which lack the gene, A␤ efflux was significantly delayed/decreased. Moreover, when A␤ is injected directly into blood vessels, rate of A␤ metabolism in blood was not different

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in the mutant mice and wild-type mice. Interestingly, the mutant mice showed lower plasma A␤ level than wild-type mice. Conclusions: Although exact mechanisms are not known, low-density lipoprotein receptorrelated protein (LRP1), P-glycoprotein (P-gp) and IGF-1 participate in A␤ removal from brain. Our data indicate that there might be other genes enrolling in the A␤ transport system across the BBB. P1-416

ACCUMULATION OF AMYLOID-␤ PEPTIDE IN HUMAN BASAL FOREBRAIN CHOLINERGIC NEURONS OCCURS EARLY IN AGING AND ALZHEIMER’S DISEASE

Alaina T. Baker, Elana Goetz, Daphne Norvin, Changiz Geula, Northwestern University, Chicago, IL, USA. Contact e-mail: [email protected] Background: The reasons for selective vulnerability of distinct neuronal populations in Alzheimer’s disease (AD) remain unknown. We have used the basal forebrain cholinergic neurons (BFCN) which are vulnerable to pathology early in the course of AD to explore the basis of selective neuronal vulnerability. Given that age is the primary risk factor for AD, we have suggested that age-related events are likely to contribute to the vulnerability of the BFCN. We and others have shown early accumulation of abnormally phosphorylated tau within the BFCN in the course of aging and AD. Objective: In the present experiments, we examined accumulation of A␤ within the human BFCN. Methods: Quantitative optical density analysis of immunohistochemically stained basal forebrains was employed in 9 normal young (20-61 years), 5 normal aged (73-83 years), and 8 AD cases (age 72-88). Two polyclonal antibodies against A␤ (1282 and B7), the monoclonal anti-A␤ 1-16 (6E10) and polyclonal anti-A␤42 and anti-A␤40 (both from Biosource) were used. Results: Robust A␤ immunoreactivity was detected within the human BFCN in young individuals regardless of age. 6E10-, 1282- and B7-positive BFCN were present in young, old and AD cases. No significant differences were observed in 6E10 immunoreactivity within BFCN of the three groups of subjects. Staining for A␤40 was very faint in human BFCN while stronger staining was observed for A␤42. A 51% increase was detected in A␤42 immunoreactivity in BFCN of old individuals when compared with the young and a 41% decrease was detected in AD BFCN when compared with the old. These differences approached significance (p⫽.0624). A high positive correlation was observed between A␤42 immunoreactivity and age in normal subjects (r ⫽ 0.72, p⬍0.02). Conclusions: These observations indicate that selective accumulation of A␤ within the human BFCN occurs early in the course of life and continues in old age and AD. Decreased A␤42 immunoreactivity in AD indicates that BFCN loss occurs preferentially among neurons with the highest accumulation of A␤. The very early accumulation of A␤ within the BFCN is likely to contribute to the loss of these neurons in AD and to cause synaptic abnormalities through release at cholinergic terminals. P1-417

HOW DOES ELIMINATION OF TNF RECEPTOR AFFECT AMYLOID-␤ PROTEIN GENERATION AND PREVENT LEARNING AND MEMORY DEFICITS IN TRANSGENIC ALZHEIMER’S MICE?

Yong Shen1, Ping He1, Jason Zhong1, Kristina Lindholm1, Gina Arnold1, Wendy Lee1, Gina Ciavarella1, Cynthia Lemere2, Matthias Staufenbiel3, Rena Li1, 1Sun Health Research Institute, Sun City, AZ, USA; 2Brigham and Women’s Hospital, Boston, MA, USA; 3 Novartice, Basel, Switzerland. Contact e-mail: [email protected] Background: Tumor necrosis factor type 1 death receptor (TNFR1) belongs to a subgroup of the TNFR superfamily, contains a cytoplasmic death domain. When TNFR1 binds its ligand (TNF␣), the ligand-receptor complex triggers apoptotic pathways by recruiting TNFR-associated death domain protein (TRADD) and/or Fas-associated death domain protein/ mediator of receptor-induced toxicity (FADD/Mort-1), two intracellular