Aging, oxidative stress, molecular chaperones, and Alzheimer's disease

Poster Presentation:

Sl86

ISOLATION AND CHARACTERIZATION BINDING PROTEIN Koichi

lijimu.

Kunnr

Ando,

Yutnkn

Kirino,

Toshihunr

OF A NOVEL

Suzuki.

Univ

of Tokvo.

APP

Tokyo

Jflpiln

Amyloid precursor protein (APP) is a cell surface glycoprotein with a large extracellular amino-terminal domain, a single transmenbrane segment, and a short cytoplasmic tail. Because of its receptor-like Structure, it is thought that the short cytoplasmic domain plays an important role in physiological functions of APP. Recently, we reproted that APP is phosphorylated at Thr668 residue (numbering for APP695 iaoform) in neurons by Cdk5 (cyclin-dependent kinase 5), the phosphorylated APP localizes mainly in neuritea, and this phosphorylation plays an important role in neurite extension in PC12 cells. These results suggest that Thr668 residue have a key to elucidate functions of APP during neuronal development. Moreover, it is repotted that the sequence of Thr668-Pro-Glu-Glu is a type I reverse turn. So, we suppose that sequence TPEE is a candidate for protein-protein interaction domain, and it may be possible that Thr668 phosphorylation regulates the protein binding affinity. To investigate this hypothesis, we searched for binding proteins which recoginize TPEE motif using the yeast two-hybrid system. We isolated a novel protein that mteracts with TPEE motif from human featal brain cDNA library, and its association is decreased when Thr668 is replaced to Ala. We will discuss the character and the possible functions of the protein that associates with TPEE motif of APP. Understanding proteins that interact with cytoplasmic domain of APP may contribute to elucidate the physiological functions of APP and pathobiology of Alrheimer’s disease.

HUMAN APOLIPOPROTEIN )844) FUNCTION IN SUPPORTING

E4 HAS A GAIN-OF-NEGATIVEMOSSY FIBER AXON SPROUTING HIPPOCAMPAL SLICE CULTURE

IN ORGANOTYPIC Hnrcr Alan Unw,

Teter.

Chnpel

Washington Hills,

UCLA,

North

D Roses. Glum Hill. Univ.

Hills.

Reseurch NC;

CA; John R Gilberr,

and Development.

Dou,q Galasko,

St. Louis.

MO:

Greg

UC Sun Diego, M Cole,

Duke

RexTr.

Univ,

Chupel

Pk.. NC;

Pu-Ting

Sun Diego,

UCLA

und

Hill,

NC:

Xu, Duke

CA; Guojun

VAMC

GRECC,

Bu,

Norfh

CA

The function of apohpoprotein E (ApoE) in neuronal sprouting may contribute to its Isotype-specific ri\k for Alrheimer’s disease. The in vitro organotypic hippocampal slice culture (OHSC) system shows granule cell mossy fiber axon sprouting in response to deafferentatton of the entorhinal cortex, a\ revealed by quantitative Timm’s staining. OHSC from ApoE knockout mouse shows defective sprouting which was completely rescued by expression of a human ApoE transgene, while ApoE tran\gene expre\\ion was only 58% as effective as ApoE3. While increased expression of ApoE rewlted in rncrrosrd sprouting, increased expression of ApoEJ &creasrd sprouting. This result suggests that the defective sprouting supported by ApoE is a gain-of-negatwe-function. Mossy fiber sprouting was inhibited by the ApoE receptor antagonist RAP, implicating a receptor-mediated mechanism. These stud& may facilitate pharmacogenetic optimization of therapeutics that target or modulate human ApoE expression levels.

AGING, OXIDATIVE STRESS, MOLECULAR AND ALZHEIMER’S DISEASE. DmrrrvGoldgrrber,

Sture Univ

oj Nen’ York, Stem

Brook.

CHAPERONES,

NY

The major risk factor for AD is agmg. Even patients who inherit one of the three known genes (APP, PSl, or PS.2) develop AD later in life. Three recent important lindings suggest a new conceptual model about molecular events leading to the generation of AP42. the main component of amyloid in AD. The first finding is a recent break-through demonstrating a direct correlation between aging and oxidative cellular stress, which causes accumulation of unfolded proteins in the endoplasmic reticulum (ER). The second finding is that mutations in PSl interfere with normal cellular response to oxidative stress by suppression of the key ER molecular chaperone, BiP/GRP78. The third finding comes from current studies in my laboratory demonstrating that APP interacts with the key molecular chaperones. BiP/GRP78, calnexin, and calreticulin, which are responsible for the proper folding and quality control of proteins in the ER. Taken together, these major findings suggest that in AD, the interaction of APP with ER molecular chaperones is the point of convergence of oxidative stress, PS mutations, and mutations in APP. I hypothesize that oxidative stress, mutations in APP, and mutations in presenilins, affect the interaction of APP with ER molecular chaperones and result in the generation of increased quantities of AP42. More specifically, I hypothesize that: (I) oxidative cellular stress leads to the unfolding and accumulation of unfolded APP in the ER; (2) PS mutations lead to accumulation of unfolded APP in the ER by interfering with unfolded-protein response to oxidative stres\: (3) mutetions in APP interfere with the

Molecular

and Cellular Biology III

proper folding of APP. In all these cases unfolded, misfolded, or mutated APP is recognized by the quality-control system in the ER and reverse translocated to the cytorol where it is degraded by the ubiquitin-proteasome degradation pathway. Finally, I hypothesize that (4) AP42 is a by-product of APP degradation in the ER. Experimental evidence supporting this conceptual model will be presented.

LOCALIZATION OF COMPLEXES OF PRESENILIN pFii?TJ APP C-TERMINAL FRAGMENTS C83 AND C99 TO CELLULAR W&zing

Xia,

William

SITES OF APGENERATION

Harvard

Med

J Ruy, Washington

T&t

Rahmati,

Michael Alison

Brigham

Harvard

Med

Sch and

Brigham

and

Univ Sch of Medicine, and

S Wolfe, Harvard M Gonte,

AND THE THE SUB-

Womm’s

Hasp,

Boston,

Med Sch and Brigham

Washington

and

Women’s

Hosp.

MA;

W. Taylor

St. Louis,

Hasp,

Boston,

Boston,

MA;

Beth L Ostuszewski,

and Women’s

Univ Sch of Medicine,

Sch und Brigham

Women’s

SI. Louis, MO;

Hasp,

MO;

Kimberly,

Boston,

Dennis

MA;

J Selkoe,

MA

Recent tindmgs indicate that presenihna (PS), with two conserved aapartate, in transmembrane (TM) domains 6 and 7, are absolutely required for Approduction by y-vxretase. Here we report that the APP C-terminal fragments, C83 and C99, can be co-immunoprecipitated with both PSI and PS2. The complexes accumulate in cells when y-secretase is inactivated either pharmacologically or by mutating the PS aspartates. We performed density gradient fractionation of isolated microsomes from cells expressing endogenous levels of PS and found that these complexes could be detected in Golgi- and TGN-rich subcellular compartments. In contrast, complexes of PSI with APP holoprotein, which is not the immediate substrate of y-secretase. occurred earlier in ER-rich vesicles. The major portion of intracellular APat steady-state was found in the same Golgi/TGN-rich vesicles, and A~levels in these fractions were markedly reduced when either PSl TM aspartate was mutated to alanine. Moreover, we used a cell-free reaction to show that these same vesicles mediate de nova generation of APand that the two TM aspartates of PSI arerequired for the new generation of A@. Thus, our results demonstrate that PSI and PS2 bind to the actual y-secretase substrates at sites of A~generation, providing compelling evidence of a direct role for presenilin\ in the final proteolytic cleavage of APP to generate AP.

EFFECTS OF PRESENILIN MUTATIONS ON vSECRETASE )8471 CLEAVAGE AND TURNOVER OF THE AMYLOID P-PROTEIN PRECURSOR C-TERMINAL Eun~u Pack-Chung, Brigham town, Taxi.

Ismx

and Wrmen’.s

MA;

Kevin

Tar-Wan

Cheng, MA Cm Hasp,

M Frlsenstein, Kim,

MA

Boston,

FRAGMENT Hasp, Churlestown,

MA:

Bristol-Myers

Gen Hasp,

Andrew,

S Yoo, MA

Squibb.

Charlestown,

MA:

Michael

Gen Hosp.

Wa//in,Qord.

S Wolfe. Charles-

CT; Rudolph

E

MA

Recent studies have revealed that presenilin I (PSI) (and perhaps PS2) 1s required for the y-secretase cleavage of amyloid P-protein precursor (APP). In the present study. we studied the effects of FAD-associated presenilin mutations (i.e. M146L, Ml46V, AE9 for PSl and N1411 for PS2) as well as presenilin loss-of-function uansmembrane (TM) aspanate mutations (i.e. D257A and D385A for PSI, and D263A and D366A for PS2) on the turnover and y-secretase-mediated cleavage of APP. Accumulating evidence indicates that the metabolic balance between turnover rate and y-secretase cleavage activity dictates the steady-state levels of both u- and P-secretase-derived APP C-terminal fragments (CTF’s). To determine the role of FAD mutations m turnover and y-secretase cleavage of APP-CTF, we treated cells with the proteasome inhibitor MG132 to selectively block the turnover of APP-CTF in the presence or absence of two separate y-secretase inhibitors. We found that MCI32 treatment leads to the increased accumulation of APP-CTF in SYSY and CHO cell lines. Interestingly, in response to the MG132 treatment, the amounts of APP CTF that accumulate were significantly and universally reduced in cells stably-expressing FAD mutant versions of PSI or PS2, as compared to wild-type presenilin-expressing cells or vector-transfected cells. Combined treatment of a y-secretase inhibitor together with MC132 gave rise to a similar degree of accumulation in wild-type and mutant cells, suggesting that reduced accumulation of APP-CTF in the MGl32-treated FAD mutant cells is likely due to the enhanced y-secretase cleavage in the mutant cells. Furthermore, in cells expressing TM aspartate mutant PSl, both MGl32 and y-secretase inhibitors had no effects on the steady-state levels of the APP-CTF (and subsequent generation of AP), implying that the TM aspartate mutation impairs both turnover and y-secretase activity. These findings suggest that FAD-associated mutations directly enhance y-secretase cleavage. but do not affect substrate availability or turnover of the APP-CTF.