ICAD - Abstract Submission P3: Tuesday, July 19, 2011 Poster Presentations P3S578

impairments, suggesting that deficits in neurogenesis may play a role in the

disease or contribute to neuropathology. To address this, in this study we ex-

amined possible molecular link(s) between neurogenic signaling and FAD

proteins.Methods:To determine the significance of APP cleavage by a-sec-

retase and the role of sAPPa in adult neurogenesis we inhibited a-secretase

activity in neural progenitor cells using metalloproteinase inhibitor. To de-

termine the role of PS1 in adult neurogenesis we developed a siRNA-ex-

pressing lentiviral vector system for the silencing of PS1 in the

neurogenic microenvironments in the adult mouse brain. Results: We

show that inhibition of a-secretase decreases neural progenitor cell prolifer-

ation without affecting their differentiation, and that this decrease can be

rescued by sAPPa. In addition, sAPPa can rescue proliferation deficits of

neural progenitor cells derived from APP knockout mice. We further

show that knocking down PS1 expression in the neurogenic niches of the

adult brain enhances cell cycle exit of neural progenitor cell and induces

their differentiation. Neural progenitor cells expressing reduced levels of

PS1 could differentiate into neurons and glia. Conclusions: This study sug-

gests that sAPPa and PS1 regulate neural progenitor cell maintenance in the

adult brain. The results of these studies imply that FAD-linked mutations

may alter neurogenic processes in Alzheimer’s disease.

P3-192 g-SECRETASE COMPOSITION AND

CELLULAR TRAFFICKING

Xavier Meckler1, Frederic Checler2, 1Institut de Pharmacologie

Mol�eculaire et Cellulaire and Institut de NeuroM�edecine Mol�eculaire,

UMR60, Valbonne, France; 2Institut de Pharmacologie Mol�eculaire et

Cellulaire / CNRS UMR 6097, Valbonne, France.

Background: Gamma-secretase catalyses the ultimate cleavage leading to

the production of amyloid peptides found accumulated in senile plaques

in the brain of Alzheimer’s disease (AD) patients. Gamma-secretase is an

enzymatic complex containing presenilin, nicastrin, APH1 and PEN2.

Within these four core components, presenilin is encoded by two genes,

and APH1 by two (human) or three (rodent) genes that can be alternatively

spliced. Also several sub complexes differing by their presenilin and APH1

contents were identified and could coexist within a cell. Whether these differ-

ent sub complexes travel to similar or distinct sub cellular compartments is still

unknown. Methods: We developed recombinant gamma-secretase sub units

tagged with various epitopes in order to follow gamma-secretase trafficking

by biochemical and immunocytochemical approaches. Results: The sub cel-

lular trafficking of gamma-secretase complexes differing by their presenilin/

APH1 contents will be presented. Conclusions: Knowing the role that

gamma-secretase composition plays in its sub-cellular localization can pro-

vide important insights into amyloidogenic processing of APP and AD.

This work is supported by the French National Foundation for Alzheimer’s

Disease and Related Disorders.

P3-193 INTERACTION OF P24ALPHA2 WITH

GAMMA-SECRETASE COMPLEX

ATTENUATES GAMMA-CLEAVAGE

OFAPP

Masaki Nishimura1, Hiroshi Hasegawa1, Lei Liu1, 1Shiga University of

Medical Science, Shiga, Japan.

Background:Gamma-Secretase mediates intramembranous gamma-cleav-

age and epsilon-cleavage of beta-amyloid precursor protein (APP) to liber-

ate beta-amyloid peptide (Abeta) and APP intracellular domain (AICD)

respectively from the membrane. Although the regulatory mechanism of

gamma-secretase cleavage remains unresolved, a member of the p24 cargo

protein family, named p24delta1 or TMP21, has been identified as an activ-

ity-modulating component. The p24 family proteins are divided into four

subfamilies (alpha, beta, gamma and delta). In contrast to p24delta1,

p24beta1 has reportedly no effect on gamma-cleavage.Methods:Using cul-

tured cells, we determined whether p24alpha2, p24gamma3 or p24gamma4

modulates APP processing. After over expression or knockdown of p24

family proteins, secreted Abeta was measured with ELISA. Protein interac-

tion was evaluated by co-immunoprecipitation assays. Mutant p24 cDNAs

were generated by PCR-based, site-directed mutagenesis. Results: Knock-

down of cellular p24alpha2 induced a significant increase in Abeta genera-

tion but not in AICD production in cell-based and cell-free assays, whereas

p24alpha2 over expression suppressed Abeta secretion. By contrast, Abeta

secretion was not altered by p24gamma3 or p24gamma4 knockdown. En-

dogenous p24alpha2 co-immunoprecipitated with core components of the

gamma-secretase complex, and the anti-p24alpha2 immunoprecipitate ex-

hibited gamma-secretase activity. Mutational disruption of the conserved di-

lysine ER-retrieval motifs of p24alpha2 and p24delta1 perturbed inhibition

of gamma-cleavage. Simultaneous knockdown, or co-over expression, of

these proteins had no additive or synergistic effect on Abeta generation.

Conclusions: Our findings suggest that dilysine ER-retrieval signal-con-

taining p24 proteins, p24alpha2 and p24delta1, bind with gamma-secretase

complexes and collaborate in attenuating gamma-cleavage of APP.

P3-194 ALCADEIN EPSILON-CLEAVAGE

FOLLOWED BY GAMMA-CLEAVAGE

Yi Piao1, Saori Hata1, Toshiharu Suzuki1, 1Hokkaido University, Sapporo,

Japan.

Background: Alcadeins (Alcs) constitute a family of neural type-I mem-

brane proteins designated Alc alpha, Alc beta and Alc gamma (J. Biol.

Chem. [2003] 278, 49448). Alcs and APP are predominantly expressed in

neuron and they largely colocalize in the brain. Moreover Alcs and APP

are cleaved in similar manner (J. Biol. Chem. [2004] 279, 24343). Alcs

are cleaved primarily by APP alpha-secretase at jaxtamembrane region

and secondarily by gamma-secretase at intramembrane region, thereby gen-

erating the ectodomain fragments (sAlc), APP p3-like fragments (p3-Alc)

and the intracellular domain fragments (AlcICD). Non-aggregative p3-

Alc was detected into extracellular milieu, and variant p3-Alc C termini

are modulated by familial AD (FAD)-linked presenilin1 (PS1) mutations

(J. Biol. Chem. [2009] 284, 36024). Methods: Plasmid encoding AlcCTF,

the C-terminal fragment of Alc generated by the primary cleavage, was

transfected in HEK293 cells expressing wild type PS1 or FAD-linked mu-

tants. The p3-Alc peptides were recovered from conditioned media and Al-

cICD fragments were recovered from cytoplasmic fraction of cells by using

specific antibodies. The immunoprecipitates were analyzed by MALDI

TOF/MS for the identification of gamma-cleaved site of p3-Alc and epsi-

lon-cleaved site of AlcICD. The minor/major ratios of p3-Alc and AlcICD

were compared for the analysis of gamma-secretase cleavage of Alcs.

APPCTFb was used as a control substrate. Results: In APP gamma-secre-

tase cleavage, the changes of minor epsilon-cleavage are reflected to the

amount of minor gamma-cleaved products. In contrast to this, the changes

of Alc epsilon-cleavage in cells expressing PS1 mutants are not always ap-

peared in the altered gamma-cleavage as an endophenotype. Conclusions:

Our observation suggests that one epsilon-site does not always determine

one gamma-site and sometimes directs to another gamma-cleaving site on

substrates. Our finding sheds light on the molecular mechanism how

gamma-secretase cleaves substrate proteins.

P3-195 g-SECRETASE CONFORMATIONAL

CHANGES IN THE ALZHEIMER’S

DISEASE BRAIN

Lara Wahlster1, Muriel Arimon1, Navine Nasser-Ghodsi1,

Alberto Serrano-Pozo1, Bradley Hyman1, Oksana Berezovska1,1Massachusetts General Hospital / Harvard Medical School, Charlestown,

Massachusetts, United States.

Background:G-secretase is the complex responsible for the final step in

the processing of the amyloid precursor protein (APP), liberating amy-

loid-b (Ab) peptides of various lengths. Longer Ab species are impli-

cated in the pathological cascade leading to Alzheimer’s disease (AD).

Presenilin1 (PS1) is the catalytic subunit of the g-secretase complex.

More than 160 mutations in PS1 have been linked to familial AD

(fAD). We have previously shown that several fAD PS1 mutations

lead to a change in PS1 conformation, affect PS1 alignment with the

APP substrate, and alter precision of the PS1/g-secretase cleavage