T I E ROLE OF ENERGY METABOLISM IN THE PATHOGENESIS

OF ALZHElMER'S DISEASE

by

RICHARD R CLOUGH

A Thesis Submitted to the Faculty of Graduate Studies in Partial

Fulfilment of the Requirements for the Degree of

MASTER OF SCIENCE

Department of Biochemistry and Molecula. Biology

Fa~ulty of Medicine

University of Manitoba, Winnipeg, Manitoba, Caaada

O August, 1999

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The Role of Energy Metabdhm in the Pathogenesb of Alzheimer's Disease

BY

Richard R Clough

A Pncticum submitted to the Facalty of Griduste Studies of The University

of Manitoba in pua . l fWWment of the requirements of the dcgree

of

MASTER OF SCIENCE

RICHARD R CLOUGH01999

Permission has been granted to the Library of The University of Manitoba to lend or sel1 copies of this thesis/practicum, to the Nationai L i b n y of Canada to micmtiùn this thesis and to lend or seii copier of the film, and to Dissertations Abstricts 1nternrtion.l to publish an abstract of t h i s thesidprrrcticum.

The author resewes other pubîieriiion rights, and neitber this thtsidpncticam w r extensive extracts fkom it MY be printed or othemise reproduced without the author's writttn permission.

1 wouid Like to thank my supervisor, Dr. Francis Amara, for allowing me to

participate in such an exciting field of tesearch, and for creating an environment tbat

demandeci independent thought and action.

Thaoks also to my thesis advisors, Dr. G. Arthur and Dr. 1. Dixon, for their expert

guidance on demie and personai matters.

M y appreciation to fellow student Binhua Liang for bis support and

encouragement over the last two Yeats.

I extend special thanks to my wife, Kathleen, for her invaluable expertise in the

preparation of this document, and for her support and advice during the lest three years.

To my daughters, Cdline and Rebecfa: Thank you for being my fan club.

TABLE OF CONTENTS

ABSTRACT ......................................................... v

LISTOFFIGURES ................................................. vii

... LISTOFTABLES .................................................. viu

LIST OF ABBREVLATIONS .......................................... ix

1.0 REVIEWOFLITERATüRE ..................................... 1

1 . I Histopathology of Alzheimer's Disease ........................ 1

1.2 Genetic Factors .......................................... 3

1 .3.0 Amyloid Precutsor Protein .................................. 5

........................... 1.3.1 Suspected hctions of APP 5

.................................. 1.3.2 ProcessingofAPP 6

............................ 1 .4.0 Pathophysiology o f APP and PAP 8

1.4.1 Evidence supporthg PAP pathogenicity ................. 9

1 .4.2 Pathogenic mechanisms o f PAP ...................... 10

1.5 Bioenergetic D e f m ..................................... 13

1.6 COX Deficit ............................................ 15

.................................. 1.7 Overexpression of APP - 2 1

1.8 OxidativeStress ......................................... 22

............................................. 1.9 Apoptosis -26

2.0 A l M AND HYPOTHESIS ...................................... - 3 1

3.0 MATERLUS AND METHODS ................................. - 3 2

line ............................................. 61

3.3 -8 Assay of glutathione peroxidase activity in APP-transfected

............................ neuroblastornacell Iiae - 6 2

3-39 Assay of supeioxide dismutase activity in APP-transfected

............................ neuroblastoma ceii lhe - 6 2

3.4.0 Classic In Vitro Mitochondrial ïmport Assay ................. -63

3.4.1 TnT in v iho transcription-transIation system ............ -63

........................ 3.4.2 Isolation of rat mitochondria -64

3.4.3 In vitro mitochoadrial import assay .................... 65

..... 3.5 Treatment of Neutoblastoma Cells with MitoTracker Probes -66

3.6.1 Immumelectron microscopy to detect APP in marnmaiian

tissue ........................................... 67

3.6.2 immuwelectron microscopy to detect APP and PAP in human ............................................ brain 68

................................................... 4.0 RESULTS 69

............................ 4.1 Yeast Two-Hybrid Technology -71

4.2 Analysis of Positive Clones ............................... -73

4.3 Vdcat ion of specificity of APPICOX 1 interaction ........... -77

4.4 Yeast Two-hybrid Assay With APP Deletion Coastnicts ........ - 8 2

4.5.0 APP and Mitochondria ................................... -83

4.51 Mitochondrial targeting signai ....................... -83

4.6.0 Assays on Neiuoblastoma C e h Overexpressing APP . . . . . . . . . . -84

4.6.1 Assays of COX and Compiex 1 eozymatic activities . . . . . . -85

4.6.3 APP overexpcession and apoptosis in neuroblastoma c e U Line

................................................ 88

4.6.4 APP overexpression and ROS production in nemblastoma ceIl

line ............................................. 88

4.6.5 APP overexpression and the formation of Iipid peroxidaîion -90

4.6.6 Anti-oxidant enzyme assays . . . . . . . . . . . . . . . . . . . . . . . . . -90

4.7.0 Evidence for the Physiological Location of APP in Mitochondria . -93

4.7.1 Classic in vitro mitochondrial import assay . . . . . . . . . . . . . - 9 3

4.7.2 Dual coior in vivo fluorescence mimscopy of neuroblastoma

ceIline .......................................... %

4.7.3 Immunoelectron rnicmscopy to detect endogenous APP and

P A P in mammalian tissue . . . . . . . . . . . . . . . . . . . . . . . . . . 103

4.7.4 Immunoelectron microscopy to detect endogenous APP and

PAP in AD and control braias . . . . . . . . . . . . . . . . . . . . . . . 105

5.0 DISCUSSION ............................................... 108

6.0 REFERENCES .............t................................ 117

ABSTRACT

In Alzheimer's disease, neuronal degeneration due to apoptosis has been liaked

to overexpression and aberrant amyloidogenic pfoçessing of the amyloid precursor

protein (APP), as well as to bioenergetic defects characterized by deficits in the electmn

transport chain's cytochrome c oxidase (COX) which leads to the generaîion of cadical

oxygen species. Several cellular mechanisrus underlying the bioewrgetic defects have

been propose& Inhibition of oxidative eneigy metabolism iacreased the emyloidogenic

processing of APP, *ch led to neurotoxicity. Overexpression of APP in cultured

normal human muscle fibres c a d a specific decrease in COX activity, followed by

ultrastructural abnormaiities of mitochondria Thus, it appears that energy metabolism

and APP processing are closely Linked. However, the mechanisms underlying this

relationship are unclear. We have demoostrated, using the yeast two-hybrid system, tbat

APP interacts with COX subunit 1 (COX I), and that this interaction involves the

extracellular domain of APP. This suggests a direct interaction between the two proteh,

and that APP may be locaüzed to mitochondria We demonstrated that APP caused

specific deficits in COX activity, induced the production of radical oxygen species and

lipid peroxidation, and exhmceâ neuronal apoptosis. However, anti-oxidant enzyme

activity was variable, and probably represented a compensatory reaction to elevated

oxygen radicals. To directly interact with COX, APP must be targeted to, and localizad

in, mitochoadna This possibility was testeci with an in viho mitochondrial import assay

which showed that APP is transported to mitochondria in an energy-dependent manner.

Furthermoie, d d fluorescence mimscopy with chimeric APP-green fluorescent protein,

and immunoelectron microscopy, dernonstrated the presence of endogenous APP and

P-amyloid peptide (the neraotoxic cleavage product of APP) in mitochondria of post-

mortem human brain tissue, with increased levels of p-amyloid peptide in AD brahs

compared to non-AD btains. Cimiulatively, the data supports a mode1 of the direct

involvement of APP and its potentially amyloidogenic derivatives in the generalized

bioenergetic deficit. This then leads to the generation of increased radical oxygen species

and subsequently, neuronal apoptois, in a seifperpeniating cycle of mitochondrial and

cellular degeneration involved in the pathophysiology of Alzheimer's disease.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 1 1

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

LIST OF FIGURES

Schematic representation of the human p-amyloid precmr pmtein(APP)

..................................................... 2

............. Schematic of pGEM9zfresûiciion map and MCS - 3 3

Schematic of pGEM5zf restriction map and MCS ............. - 3 5

Schematic of pAS2-1 restriction map and MCS ............... - 3 9

.................... Schematic ofhuman APP- construct -41

Schematic diagram of the MATCHMAICER GALA-based two-hybrid

systems ................................................ 72

Screening an AD fusion library for proteins that interact with a bait

protein ................................................ 74

Matchmaker two-hybrid screen ............................ - 7 5

Sequence of clone A-1 4; the partial COX 1-~RNA* gene ......... 76

Restriction enzyme digestion of pAS2-l/APP-, with EcoR 1 ... 80

Sac 1 restriction enzyme digestion of pAS2-l/APPf70/Nf70/N'171020 constnrt - 8 1

Hhd III restriction of coostnicts pAS2.1/APPm.no, & pAS2-

........................................... P m 83a

Targeting APP into mitochondria potentiates apptosis ........... 89

Induction of ROS and lipid peroxidation in SIC-N-SH cens ........ -91

Cornparison of the fke radical scavenging enzyme activities ...... 94

Mitochondrial import in vitro ............................... 97

EcoR 1 and BamH 1 restriction of pEGFP-Nl/APP693/N/Nf ,, ügation

vii

............................................. constnicts 100

Figure 18 EcoR 1 restriction of pEGFP-NI and pEGFP.Nl/APP,,, ..... 101

..................... Figure 19 Pst 1 restriction of pCMV/myc/mito/GFP 102

Figure 20 SK-N-SH nemblastoma ceîi mitochoadrial labelling ........... 104

Figure 2 1 Localizationof APP by immunoeIectrunmi~t~scopy in mammalian brah

andkidaey ............................................. 106

Figure 22 -zation ofAPP and PAP by immmoelectron mic~oscopy in human

.................................... ADandnon-ADbrain 107

LIST OF TABLES

Table 1 Interdon of APP and COX 1 in yeast ...................... -78

Table 2 Complex 1 and cornplex TV enzyme activities in neuroblastoma ceils

overexpressbgAPP ...................................... 87

8OHdG

AA

ActD

ADDL

AGEP

AIF

*P

apo-E

BD

CAA

ch

Ci

CIAP

CMR(gic)

COQ

CPA

cyh

DCF

DCF-DA

AiCr

ER

LIST OF ABBREVIATIONS

8-hydroxy-2'-deoxy~osiw

arachidonic acid

activation domsin

kta-amyloid peptide-derived difiible ligand

advanced glycation md poduct

apoptosis induckg fpctor

ampicillin

aplipoprotein E

binding domain

cerebral amyloid angiopathy

colony fonning units

curie

calfintestinal alkaline phosphatase

cortical and cerebellar glucose metabolic rate

coenzyme Q

cis-parinaric acid

cycloheximide

dichloro fluoroscin

dichlorofluoroscein diacetate

electrochemical gradient, or proton motive force

endoplasmic reticdum

FAD

GSase

GT

HNE

kan

LTP

MF0

m

NMDA

NP

PO

PCLAA

PHF

PS

PTP

RAGE

RE

ROI

sAPP

SR

TMD

kanamycin

long term potentiaîion

mixeci fùndon oxidation

neurofibrillary tandes

N-methyl-D-aspartate

neuritic (or senile) plaque

mitochoadria-deficient

phenoVchlomform/isoamyl alwhol

paired helicai filaments

p r e s e n . protein

permeability transition pore

receptor for dvanced g l y d o n end products

restriction endonuclease

reactive oxygen intermediate

secretory (or soluble) APP

scavenger receptor

trammembrane domain

1.0 REVIEW OF LITERATURE

Alzheimer's disease (AD) is a cerebral degenerative disorder clwiracterized by

graduai loss of memory, reasoning, orientation and judgment afExtiog 1% of the

population of the western world 1 %6), and S-Iû% of ail people over 65 years

ofage (Hurtley, 1998). Symptoms are correlated with the appeanuice of amyloid deposits

rnainty in the hippocampus and association cortex of the brain (Roth et d., 1966;

Goedert, 1993; Haas and Selkoe, 1993; Kosik, 1994; Trowjanowski and Lee, 1994). The

brains of AD patients contain many extraceliular, amyloid neuritic plaques (NP), which

are dense, heterogeneous, extracellular deposits, largeiy composed of the 3943 amino

acid beta-amyloid peptides (PAP), the cytotoxic cleavage products of the betaamyloid

precusor protein (APP) (Figure 1). AD brains also contain neurofibriiiary tangles (NFT;

intracellular accumulation of a b n o d y phosphory lated mictotubuie-associated protein

tau, arrangeci into paired helicai filaments [PH7]), dystrophie neUrites (enlarged neuronal

processes nIled with PHFs), and show profound synaptic loss and giiosis (activateci

microglia and reactive astrocytes) (Glenner andWong, L 984 aand b; Masters et al., 1985

a and b; Masters and Bayreuther, 1988; Games et aï., 1995; Lamb, 1997).

The classic NP coasists of a cent . $AP-immunoreactive amyloid core

surrounded by dystrophie neurites (Cummiags et d, 1998). Amyloid is deposited in

cerebral blood vessels (cerebtal amyloid angiopahy [CAA]) as well as in diffuse and

Figure 1 Schematic representation of the human Pamyloid precrnsor protein (APP). Various isofonns of APP are produced by alternative splicing of pre-mRNAs to exclude the 0x02 antigen domain (amiao ôcids 345-364 of APP,) to produce APP,5,, the Kunih-type prokase-inhibitor domain (amino acids 389-344 of APP,) to produce APP,*,, or both domains to produce AFP, a-secretase-mediateci cleavage of a proportion of cellular APP molecules at ~ y s q ~ e u ~ ' of APP, (PAP Lys"%eul') precludes the formation of PAP. Intracellular processing of APP by P- and y-sexetases, however, yields intact PAP, wnsisting of 28 amiw acids derived h m the extracellular domain and 12-14 amino acids h m the transmembtane domain. A number of point mutations in the APP molecule have been identifiecl in the vicinity of the PAP sequence. These mutations CO-segregate with family pedigrees predisposed to early onset FAD, and are characterized by alterations in the formation aad deposition of PAP. (Price et al., 1998).

neuntic plaques (Oiichney et al., 1995; Vin- et al., 1996). Newe celi los, prticdarly

affecting the larger neucons of the superficial cortex, is a consistent feature of AD

(McGeer et al., 1994; Moms, 1995; Terry, 1997; Gomez-Ma et uL, 1997). Presynaptic

terminai density is deereased by an average of 45% at the tirne of autopsy (McGeer et al.,

1994), and synapses are greatly d u c e d in the region of NPs (Terry et al., 1991).

Extranemaal ghost tangies may be seen in the entorhinal cortex and represent the

insoluble residue of neurons thst bave died (MO&, 1995).

1.2 Gcnetic Factors

AD occurs in two forms: (i) the rare, early onset familial AD (FAD), an

autosornai dominant disorder c a d by mutations in the genes encoding three integral

membrane proteins, APP, presenilinl (PSI) and presenilin 2 (PS2), whose precise

fiinctions are unknown (Goate et al., 199 1 ; Chartier-Harlin et al., 1 99 1 ; Murteii, 199 1 ;

Hendricks et al., 1992; Mdan et al., 1992; Levy-Lahard, 1995; Rogaev et al., 1995;

Shemington et al. 1995; Hardyy 1997; Price and Sisodia, 1998), and (iï) the common

sporadic, non-familial AD, in which the non-mutated forms of all three proteins are

probably involved (Dewji and Singer, 1997).

Down Synhme O S ) patients over the age of 30 almost universally have AD-

type paîhology in the brain, and the prevalence of dementia increases with age

(Wisniewskî et al., 1985). Patients with DS have three copies of chromosome 21, which

bears the gene for APP, and have i n d production of PAP (Govoni et al-, 1996;

Hardy, 1997).

Severai missense mutations of the APP gene have been described in a iimïted

number of early-onset AD families. The mutations accurmar the beginning and the eud

of the PAP sequence within the APP gene. They affect APP ptocessing in different

ways, but ail increase the production of P A P (Goate et d., 1199 1; Chartier-Hariin et al.,

199 1 ; Hardy, 1997), particularly the form with 42 amino acids (PM,-& which has the

greatest association with neurotoxicity (Mann et al., 19%; Lendon et al, 1997; Hardy9

1997; Cummings et al, 1998).

Several missense mutations of the PS 1 gene on chromosome 14 account for most

cases of FAD. These mutations also increase the production of PAP,, (Sherrington et al.,

1995; Mann et al., 19%; Hardy, 1997). A smaü number of families have mutations of

the PS2 gene on chromosome 1 (Rogaev et al., 1995; Levy-Lahard et al., 1995 a and b),

which also increases production of B A . (Mann et al., 1997).

A major genetic risk fstor for lateonset sporadic AD is possession of the apoE

€4 allele (apoE-4) (Corder et al., 1993), which is associated with i n d deposition

of PAP (primarily PM& an apparent facilitated configurational change h m diffuse to

aggregated amyloid in a beta-pleated sheet wnîiguration (GomQ-Isla et al., l9%), and

an i n d fkquency ofNPs and CAA (Gomn-Isla et al., 1996; Oiichney et al., 19%).

A mutation in the gme enooding q-macrogiobulin (qhQ also increases the susceptibility

to develop AD, possibly by allowing f3AP to accumulate to abnozmally high levels

( M m y 1998)-

There is an increased ri& for AD in oBpring of AD affected womea, suggesting

one risk factor for AD is matemal inheritance. This wdd be related to inheritance of

mitochondrial DNA (-A) @diand et ai., 19%), as it is lmown mitochondnal

fbnction is impaïred in AD.

APPs are synthesized as N- and O-giycosylated integral tninsmernbrane proteins

which span the lipid bilayer once. There is a large amiw (NHktenniaal extracellular

domain, a transmembrane domaia (TMD) and a short 47 residue Long cytoplasmic

domain (Dyrks et al., 1988; Weidemann et al., 1989) (Figure 1). Different tianscripts of

APP arise h m alternative splicing of APP mRNA, the major transcripts h a h g 695

residues (APP,,), 75 1 residues (APP,,), and 770 residues (APP,) (Kang ef al., 1987;

Tanzi et ai., 1988; Kitaguchi et al., 1988; Ponte et al., 1 988; Oltersdorf et al., 1 989; Van

Nostrand et al., 1989).The boxes above the APP schematic in Figure 1 (containing

oumbers 770 and 751) represent the amino acid sequences misshg h m N P , that

when spliced into APP, genenite the isoforms, APP,,,, and APP,5, (Rice et al., 1998).

13.1 SuspectedfiinctionsofAPP

One problem in understanding the pathophysiology of AD is that the biological

fiinctions of APP are unknown. Suspected fbnctions of membrane-bound AFP include

inhibition of extracellular serine proteases (Oltersdorfet al., 1989), inhibition of platelet

coagulation factor XIa (Smith et d., 1990), involvement in ce11 adhesion (Schubert et al.,

1989; Breen et ai., 1991; Srnall et al., 1992; J i . et al., 1994), neurite outgmwth and

extension (L,eBlanc et al., 1992; M i l 4 et al., 1992; Koo et al., 1993; Small et al.,

1 994; Jin et ai., 1994; Qiu et al., 1999, synaptic plasticity (Mattson et al-, 1993 a), and

sroaptotrophic effects (Mattson et ai., 1993 a; Mucke et d., 1994). The secreted form of

APP (sAPP), the large NH,-teRlljIial -ent produced by normal, nonamyloidogenic

pmteoiytic cleavage within the PAP sequence region by a-secretase (Figure l), sîabilizes

intrsceiiuiar k e CaZ+ ([Ca2+]& and protects neurons against excitotoxic insuits (Mattson,

1994). This suggests sAPPs are important for moduiating activitydependent processes

throughout the brain. It was shown that sAPP activated high conductance potassium (K*)

channels that caused a decrease in [Ca '+Ii (Furukawa et al., l!J%), thereby suppressiag

[Ca respll~es to the excitatory neurotransmitter, glutamate (Glu), and modulahg the

effects of Glu on neUrite outgrowth and ceLi survival (Matison et al., 1993 a; Mattson,

1994). This may explain the effects of APP on synaptogenesis (Roch et al., 1994) and

synaptic plasticity (Huber et al., 1993).

APP may fiinction as a ce11 Surface teceptor, ~ ~ ~ e ~ t e d to various intraceliular

pathways M a its cytoplasmic domain interacting with intraceildar efkctor molecules. Its

structure is similar to that ofthe extracpiiular maîrk protein receptors, the integrins, and

its carboxy (COOH)-terminal region bound an iatraceiiular protein, APP-BP 1, which

may be involved in the celiuiar ubiquitination paîhway (Chow et ai., 19%).

13.2 Proccssing of APP

A fhdamental event in the pathogenesis of AD is the enhanceci, aberrant,

amyloidogenic processing of APP, which causes excessive productionof neurotoxic PAP

species. A constitutive, non-amyloidogenic, pmteolytic cleavage of APP, by an

uncharacuerized a-sectetase, occurs in the hansalgi network or other end-stage

comparmients of the protein secretory pathway (Sambamurti et al., 1992; De Strooper

et ai., 1993; Kuentzel et al., 1993), includiag caveolae @rem et al., 1998), or at the cell

surface (Sisodia, 1992). The a-secretase generates the soluble 90-1 10 kDa sAPP (the

ectodomain of APP) a d a membranebound 9 kDa COOH-tenninaI derivative. The

sAPP COOH-terminal site of cleavage is at amino acid 16 of the PAP sequence, which

means the NH, terminus of PAP is not produced by normal non-amyloidogeoic

proteolytic cleavage (Wiedemann et al., 1989; Sisodia et al., 1990; Esch et al., 1990;

Anderson et ai., 199 1 ; Sisodia, 1992; Haas and SeUcoe, 1993).

PAPs are generated by endopmteolytic cleavage of APP (amyloidogenic

processing) by uncharacterized endopeptidases d e d B- and y-secretases, at the amino

and carboxy termini, respectively, of the PAP sequence of APP (Haas md Sekoe, 1993).

The initial step in the abnormal processing of APP in AD occurs at the NH,-terminus of

the PAP sequence, generating a potentiaiiy amyloidogenic COOH-terminal -ent

containhg the PAP sequence, the transmembrane, and cytoplasmic domains of APP

(Estus et ai., 1992) (Figure 1). It is believed this cleavage occurs in the mediai-golgi

network and endosomaVlysosomal pathways (Shoji et al., 1992; Caporas0 et ai., 1992;

Estus et al., 1 992; Golde et al., 1 992; Haas et ai., 1992; Kwps et ai., 1992; Perez et ai.,

19%; Thinakanui et al., 1996). or in the secxetory @way proximal to the cell

membrane (Busciglio et al., 1993; Haass et ai., 1993; Seubert et ai., 1993). The P-

semtase cleavage is thus the first step in Alzheimer's-type amyloidogenesis, and sets the

stage for the next cleavage by y-SecrefaSe. The latter cleaves between residues 40 and 4 1,

or resïdues 42 and 43 (cleavage between tesidues 43 and 44 can dso occur) of the

COOH-temunai fiagrnent (Kosik, 1999). The y-seciletase cleavage site requires eqmsure

by membrane damage, such as induced by lipid peroxidation @yrks et al., 1992).

Cleavage of the COOH-terminal amyloid fhgments by y-secretases to reiease the 39-43

amino acid fMP species pmôably occurs in the endosornellysosome pathway (Busciglio

et al., 1993), although it appears that the endoplasmic reticdum (ER) may be the si@ of

generation of PA& but not P A P , in nemas (Hartmann et al., 1997).

1.4.0 Pathophysiol~ of APP and PAP

The formation and slow accumulation of the cytotoxic PAP in NPs in the

hippocampus and adjoining areas of the brain, altered metabolism of APP,

overexpression of APP, r e d d glucose uptdce into brain cells, impakd oxidative

phosphorylation (OXPHOS) due to mitochondrial dysfiuiction, oxidative damage to

cellular proteins, Lipids and nucleic acids, neuronal excitotoxic mechanisms, and

apoptosis have al1 been implicated in the newodegenerative process of AD (Mattsoa,

1993[a] and 1994; Mark et al., 1995; Dewji and Singer, 19%; Keller et al., 1997;

Shoulson, 1 998).

There are numemus PAP species with extensive NH,- and COOH-terminal

heterogeneity (Murphy et al., 1999). About Wh of secreted PAPs are the soluble forty

amino acid long PAP,, and are the 42 and 43 residue BA& and P A P , which are

highly fibrillogenic, readily aggregated, and neurotoxic (Jarrett and taasbury, 1993;

Yankner, 1996 ; Mann et al., 1996 & 1997; Lansbury, 1997; Mettson, 1997). The major

PAP species in cell culture media and cerebmspinal fluid (CSF) w m found to be P M a

( 5 0 - 7 0 , although some P A P , (5-2û%) was dso present, as weil as lesser amounts of

other peptides, including P A P , (Murphy et al , 1999). Self-association occurs fader for

the longer, more hydrophobie form of PAP (gAP, versus PAPA (Jarrett et al, 1993;

Snyder et ai., 1994), it assembles more rapidly into nlaments in vitro (Jarrett et al.,

1993), and is prone to aggregate into amyloid fibrils. The amyloid fibril is the neurotoxic

form of PAP, witb the soluble and non-fibriliar aggregates mostly being non-toxic (Püre

et al., 1993; Lorenzo and Yanbier, 1994). and PAP,, peptides are deposited early

and selectively in plaques (Roher et al., 1 986; Iwatsubo et al., 1994; Lemm et al., 1 9%),

and are deposited earlier in the disease process than PAP, (Saido et al., 1995).

1.4.1 Evidence supportiig PAP pathogenicity

The production and accumulation of PAP is accepteci as king the primary event

involvecl in the pathogenesis of AD (Cordeil, 1994; Sekoe, 1994 a), and the abnomial

accumulation of PAP is thought to be due partiaily to increased generaîion of APP and

to abnomial processing of it (Sekoe, 19Wb and c). A primary pathogenic role for APP

and PAP is suggested by the fïndings that aggrrgated PAP is directly toxic to cultured

nerve cells and clonal celi lines (Yankner et al., 1989 and 1990; Koh et al., 1990; Pike

et al., 1991 and 1993; Matbonet al., 1992; Loo et al., 1993; Behl et al., 1992 and 1994),

increased accumulation of PAP,, in the brain causes amyloid nemtoxicity leading to

dementia (Yanlaier et ol., l m ) , PAP,, is more abmdant in AD brain tissue tban in age-

matched contmls (Selkoe, 1994 d), a d all FAD mutations influence APP processing in

a marner that resuits in elevated production of the highly amyloidigenic PM,, and

peptides (Price et al., 1998). AM mutations causing FAD must alter the proteolytic

pmcessing of APP, such as by altering y-secretase activity (Scheuner et al, 19%; Murphy

et al, 1999)- This rnay resuit in decreased production of the nemprotective sAPP

(Mattson et al, 1993).

In relation to the pathophysiology of AD, it is known that PAP and P-amyloid

fibrils disturb the integrify of ceil membranes, genenite ceactive oxygen intermediates

(ROI) and the accompanying cytotoxic cellular oxidant stress, incrwise msceptibility to

Glu-induced excitotoxiç stress, pmmote microglial activation and cytokine reiease, and

eventually cause neuronal cytotoxicity and cell death (Yankner et al., 1 WO; Davis et al.,

1992; Araujo and Cotman, 1992; 1993 ; Koo et al., 1 993; Pike et al., 1993; Haass and

Seikoe, 1993; Kosik, 1994; Tmwjanowski and Lee, 1994; Hensley et al., 1994; Behl et

al., 1% Kiegeris et al., 1994; Lorenu, and Yanlaier, 1994; Me& et al., 1995; Benzi

and Moretti, 1995; Co- and Anderson, 1995; Busciglio and Yankner, 1 995).

f3AP7s induction of plasma membrane iipid peroxidation, impairment of ion-

motive ATPases and Glu uptake, uncoupLing of G-protein-linked receptors, and

generation of reactive oxygen species (ROS), al1 contribute to loss of [Ca2+Ji homeostasis

tbat contributes to ce11 death (Mark et al., 19%).

One way PAP adversely affects neuronal fimctioa and survival is by its reportai

stimulation of the production of hydrogen peroxide (H203, which led to oxidative

damage and celi death (Behl et al., 1992 a d 1994). Oxygen fadicais ptoduce injuzy to

neuronal membranes and to mtDNA (Mecocci et al., 1994), make neurons more

VUlDerable to dysfimction produced by Glu (Beal, 1992), and contribute to aggregation

and deposition of @Al? (Besl1995). PAP also induces activation of microgiîa leadhg

to secretion of toxic f k e radicals and cytokines, &ch may M e r damage neurons

(Meda et al., 1995)-

Proposed mechanisms for PM'S deletenous effects include direct neurotoxicity

(Yankner et al., 1989 and lm), and induction of Ca2+ metabolism deraagements leaâing

to enhsuiced excitotoxicity (Mattson et al., 1992 and 1993 b; Mattson, 1994 a),

membrane perrneability enhancement (Simmons and Schneider, 1993)' and formation of

Ca2+ charnels that lead to abnomLally hi& cytosolic ca2+ concentration and subsequent

c d death (Arispe et al., 1993). PAP forms fke radical peptides and aggregates that

destabiiize [Ca2+Ji and make murons vulnerable to metaboiic insults (Mattson, 1994 a).

The disturbed ca2+ homeostasis and increased intraneuronal CaZ+ concentration induced

by PAP may activate intraceliular proteases, lipases, and other enzymes, leading to ce11

death (Mattson et al., 1 993 b; Hardy and Higgins, 1992; Kbachaturian, 1 994).

Senile plaques (NPs) contain a high concentration ofmicroglia that express class

A scavenger receptors (SR) (Wiewski et al-, 1989; Christie et al., l9%), and glycated

matrix proteins that are ligands for class A SRs are present in Alzheimer lesions (Smith

et al., 1994; Vitek et ai., iW). It is postulated that deposition of p-amyloid fibds

induces mimglia to adhere to p-amyloid fibds via their class A Sb, pmbably in an

attempt to clear them h m the extmceliular milieu. The high density of SR Ligands in

these PAF deposits causes immobüization of mimglia that corne into contact with hem,

and induces these microglia to Secrete cytokines, ROS and nitmgen species that injure

neighbouring neurons (El Khoury et al., 19%). It has previously been s h o w that SRs

participate in the production of ROS (Hamiag et ol, 1986).

Advanced glycation end p d u c t s (AGEP) wdd play an important role in the

etiology of AD. AGEPs produce modifieci structures that form protein cross-LiaLs, and

are known to generate ROIS (Vitak et al., 1994, Yan et al,, 1994; Ledesma et al., 1994).

Protein cross-ünking inhibits the physiological bc t ion of many proteins, and AGEPs

may contribute to the ûansfomiaton of the soluble form of PAP into its insoluble

version. Their induction of oxidative stress may also contribute to their suspecteci d e

in the pathogenicity in AD (Thorne et al., 19%).

The receptor for advmced glycation end products (RAGE) is one receptor

postdateci to mediate the interaction of SAP with endotheLial cells and murons, resuithg

in cellular oxidant stress, and with mimgiia, resuiting in ceiiular activation (cytokine

production, chemotaxis, haptotaxis). Interaction of PAP with neurons a d vascular cells

caused oxidant stress and subsequent deleterious effkcts on cellular fùnction and organic

homeostasis (Yan et al., 19%).

PAP aggregates and amyloid fibrils have also been found inside ceUs (Knawr et

al., 1992; Fuller et al., 1995; Yang et al., 1995; Tumer et al., 1996; Wdd-Bode et al.,

1997; Xia et al., 1997; Xu et d, 1997; Tierari et d-, 1997; La Ferla et al., 1 997), and

PAP an be generated in the ER (Wiid-Bode et al , 1997; Tienari et al., 1997) and be

taken up by endocytosis (Kna~% et ai., 1992; Yang et al., 1995; La Ferla et al., 1997;

Urmoneit et al., 1997). Thetefore, P A P may target intraceliular molecules (e.g. ERAB),

mediatïng cellular stress and uîtimately apoptosk, due to inaPased amounts of SAP Cyan

et al., 1997).

It has been known for some time that there are specific bioenergetic defsts in

multiple tissues in AD patients (Swerdlow et al., 1997), and that a bioenergetic

mechanism may be a primary event in the pathogenesis of AD (BIass et al., 1988; Blass

and Gibson, 199 1; Beal, 1992; Hoyer, 1993; Mutisya et ut., 1993; Swerdlow et al.,

1994). Defeets in giucose utilizitio~!~ and citric acid cycle metaboiism have been

describeci in AD (Parker and Padcs, 1995).

Abundant evidence indiates that a b n o d t i e s of cerebral metabohm are a

major factor underlying the pathogenesis of AD. Many reports have shown that

reductions in AD brain occur in the pyruvate dehydrogenase complex (the link of

glycolysis to the Kreb's cycle), the alpha-ketoglutarate dehydrogenase complex (the iïnk

of Kreb's cycle to glutamate metaôolism) and COX of the mitochondnal ETC (the Link

of the Kreb's cycle to oxygen utilizabon). Additionai d t s indicate that the reductiotls

can also be secondary to other causes including oxidative stress. A variety of data -est

that the mitochondrial iasiifociencies contribute significantly to the pathophysiology of

AD (Gibson et al., 1 W8).

A key process in AD is decread glucose turnover and a subsequently derirPased

oxidative phosphorylaîion, linked directly to secondaxy amyloid formation and nerve ce11

atrophy (i.e. disnirbances of OXPHOS and glucose tumover induce $AP production)

(Roberts et al., 1991; Meier-Ruge and hni-FreddaR, 1997). The reduced OXPHOS

causes the gewraton of PAP and the loss of neuronai synapses, because APP is not

inserted into neufonai membranes in the absence of d c i e n t ATP. This I d to the

generation of PAP and to amyloidosis (Meier-Ruge and Bertoni-Freddari, 1997).

Blood glucase levels are a b n o d y low in Alzheimer's dementia, and it was

found that the fasting plasma glucose level was signincantly r e d d (about 2W) in

patients with Alzheimer's dementiacompared to those without the disease (Itagaki et d,

1996). In vivo imaging of AD patients using positron ernission tomography (PET)

showed progressive reduction in brain glucose metabolism and blood flow in relation to

dementia severity. The reductioas reflect physiologicai down-regdation of gene

expression for glucose delivery, OXPHOS, and energy consumption in brain

(Chandrasekaran et al., 19%). In a shdy (using PET) measuring the cerebella and

cerebral metabolic rate for glucose (CMRglc) in AD and age-matched normal control

subjects, it was found that, in severe AD, both cerebeiiar and cerebral glucose

metabolism were significantly reduced (Ishii et al., 1997). Plasma protein glycation

specincaiiy derived h m giuwse was evaluated in moderate and severe AD patients and

compared with an age-matched contml group. The results suggested an eventual

impairment in glucose peripheral use or an iacrrase in protein glycation rate associatecl

with AD (Rivîere et al., 1998).

Deficits in energy m e t a b o h are a major cause of amyloidogenic processing of

APP. Inhibition of cytochrome c oxidase (COX, Complex IV) of the electron transport

chah (ETC) caused amyloidogenic prooessing of AeP (ûabuzda et al., 1993; Iverfeldt

et al., 1993). ATP depletion d e d l y induced the production of a potentially

amyloidogenic 1 1.5 kDa COOH-terminril derivative of APP in the -tory pathway

(Gabuzda et al., 1994). In COS cells, metabotic stress due to giucose deprivation uuised

a 26% decrease in soluble APP (sAPP) secretion. Sodium azide, an inhibitor of COX,

decreased sAPP release in a concentration dependent mamer (maximum -75%). These

results suggest that the inhibition of energy metabolism c m influence APP pmessing

leading to a d e d secretion of non-amyloidogenic hgments of APP (Gasparini et

al., 1 999, and an in& g e n d o n of potentially amyloidogenic h p e n t s (Gabuzda

et al., 1994).

1.6 CQX ûeficit

The mitochondrial ETC'S cytochrome c oxidase (Complex N, COX) is a-n

subunit multimeric enzyme, located in the inwr mitochondrial membrane. Only three of

its subunits (COX 1, COX II, COX III') are encoded by mtDNA (Edland et d., 1996).

COX is involved in the mitochondna generation of ATP by the p m s s of oxidative

phosphorylation (Wallace, 1999).

COX was shown to be catdytidy depressed and kinetidy abnonmi in AD

cerebral cortex, fibroblasts and platelets, suggesting the enzyme may be stmcturaiiy

altered It was, however, expiessed at normal levels (Parker et al., 1990; Kish et al.,

1992; Mutisya et al., 1993 and 1994, Parker et al., 1994 a and b; Beal, 1995; Parker and

Parks, 1995).

The COX and other ETC activities were assayed in platelet mitochondria isolated

fkom patients with AD. Five of 6 patients had süiking 5Wh reductio~~~of plateIet activity-

Other ETC cataiytic activities were not signiscxmtly cliffirent than cobttol values- It was

postulated that AD may be a systemic illness, a ptimary defect in COX may be

pathogenically important in its ptoduction, and the mitochondcial genes encoding COX

subunits may be Unportant in pducing the defect (Parker et al., 1990; Parker, 199 1). In

another assay, mean platelet COX wtivity in AD patients was sisnificatltly l e s (-1 8%)

than in contn,ls Complex Ki (ubiquino1:cytochrome c oxidoreductase), cornplex II

(succinic dehydrogenase), and ciîrate synthase were aü assayed as internai wntrols and

were not signincantly different in controls and Alzheimer patients. This points to a

relatively specinc loss of platelet COX activity in Alzheimer disease patients (Parker et

al., 1 994 a).

Fibmblasts h m AD patients have also displayed decrwised COX activity. The

results from determining the basal oxygen commption rate (403 supported the

hypothesis that subtle dyshctions of oxidative energy-producing processes are present

in fibroblasts h m sporadîc AD patients. Io tissues, such as the brain, that rely heavily

on oxidative metabolism for their hction, similar altaations may trigger molecdar

mechanisms leading to celi damage ( C d et al., 1997). The ETC activities in

mitochondria isolated h m autopsied brain samples h m AD patients and h m wntrols

with and withouî known neurologie disease danooshated a genetalized depiession of

activity of ail ETC complexes, being most marked in COX activity. These f'indings agrw

with the proposition thaî the ETC is defective in AD brain, and the defect centers about

COX (Parker et al., 1994 b ). The activity of COX in homogenates of autopsied btain

regions of patients with AD and controls showed that mean COX activity in AD brain

was reduced in hn ta i (-26%), temporal (-17%), and parietal (-160/0), though not

signifiant) cortices. The reduction of COX activity, which is tightly coupled to neuronal

metabolic activity, was pmposeâ to be explained by hypofünction of neurons, neuronal

or rnitochondrïal los , or possibly by a more primary, but region-specific, defect in the

enzyme itself. It was concluded that the absence of a COX activity reduction in ail of the

examined brain areas does not support the notion of a generalized brain COX

abnormality. Although the functiomd significance of a 16-26% cerebral cortical COX

deficit in human brain is not known, a deficiency of this key energy-metabolizing enzyme

couid reduce energy stores and thereby contribute to the brain dysfunctioa and

neurodegenerative processes in AD (Kisb et al., 1995). Parker and Parks (1995) showed

that purifieci AD brain COX displayed anornaIous kinetic behavior compared with

control brain COX, in that the low Km binding site was kinet idy unidentifiable. For

purposes of cornparison, COX was also purifiecl h m a standad beef heart preparation

and was found to display normal kinetic behavior. It was thus proposed that AD brain

COX rnay be stnicturaliy abnormal and may, in agreement with other findings, make an

important contniution to the bioenergetic defect seen in AD (Parker and Parks, 1995).

Mutisya et al. (1994) measured the activities of Complexes 1-IV in hntai, temporal,

parietal, and occipital wrtices of AD brains. Complexes 1-III showed a small decrease

in activity in the occipital cortex oniy. The most consistent change was a 25-3W

reduction of COX in four cortical regions e xamkd. Reichmam et ai. (1993)

demonstrateci a marked decrease in COX, and lesser deficits in Complex II and ïIï

activities, in homogenate and p d i e d mitochondnal fiactions from AD patients

c o r n e with controls. Another study thaî evaluated the Level of COX in various brain

regions of pst-mortem AD and control patients, found that there was reduced COX

levels and activity in ail cortical areas examined, suggesting a generaiized suppression

of oxidative metabolism throughout the cortex (Wong-Riley et al., 1997).

Alteration in mitochondrial COX gene expression might underlie the reduction

in COX activity in AD. There was found to be a selective reduction in mRNA levels for

the mitochondrialencoded COX ï ï subunit, both in regions with NFTs, sede NPs, and

nemaal loss and regions relatively spared h m these neuropathological changes. The

data suggested that the reduction in COX activity in brain regions h m individuais with

AD may be a result of an alteration in mitochondrial COX gene expression that extends

beyond neurons directly affécted by shuctural pathology (Sirnonian and Hymas 1994).

Supporting the above £indings, brains h m five patients with AD showed a 5 0 % 6 5 %

decrease in mRNA levels of COX subunits I and Di in the middle temporal association

neocortex, but not in the primary motor cortex, as compared to five control brains

(Chandraselcaran et al., 1994). Thete was also found to be a 50% decrease in mRNA

levels of mtDNA-encoded COX 1 md III in pst-mottem AD brains, in an association

neocortical region (micitemporal cortex), but not in the pr ïmq motor cortex unaffecfed

in AD. There were 5û-6û?/a decreases in mRNA levels of nuclear DNA (aDNA)-eded

COX IV and the beta-subunit of the F3,-ATP synthase in rnidtemporal cortex, but not

in the primary motor cortices of the AD braias (Chandrasekaran et al., 1997). Follow up

work demonstrated that COX II mRNA h m pst-mortem brain tissue was mafkedly

reduced in the entorhinal cortex and the hippocampd formation compared with wntrol

brains. In the hippocampus, reductioas were in regions severely affected by AD

pathology as well as in regions that were relatively spared (Chandrasakruan et al., 1998).

These data suggest that the decrease in COX 1, II and BI subunits mRNA in affecteci

brain regions may contribute to reduced braia oxidative metabolism in AD (COX is a

marker for oxidative metabotism) (Chandrasakanui et al., 1994). As weii, it appears

reduced neuronal activity and downreguiation of OXPHOS machinery cause coocdinated

decreases in expression of mitochondrial and nuclear genes in the association cortex of

AD brains (Chandrasekaran et al., 1997), and reduced rnitochondrial energy metaboüsm

reflects loss of neuronal connections (Chandrasekaran et al., 1998).

Cybrids have been used to study OXPHOS. Cybrids are formed by the

tramferring of mitochondria from living patients to mitochondria-defkient ceils (pO

cells). The resuitiag cybrids enable one to determine whether any obsewed defects in

OXPHOS in a patient's tissues are attributable to alterations in a patient's mtDNA, as the

patient's mitochondna now firnction in the presence of adsetent nuclear genome (Beal,

1997). Neuroblastoma AD cybrids demonstrated a 52% decrease in COX activity, an

elevation of ROS levels, increased basal cytosoiic [Ca2+] aod an enhanced sensitivity to

hositoi-1 A S - p h - m release. It was suggested that the subtle alterations

in Ca2+ homeostasis and ROS geneiaîion might lead to inaaised SUSCePfibility to cell

death under circumstmces not ordiaarily toxic (Sheehan et al., 1997). AD cybrids were

show to have increased levels of ROS in the presence of in& compensatory fkee

radical scavenging enzymes. It was postulated a primary biochemicai defect (COX

dysfunction) crrates a s e c o ~ biochemical defect ( i n c d ROS gewration),, bath

king relevant to neurodegenetation (Swerdlow et al., 1997; Beal, 1997).

COX may also play a aitical d e in specinc groups of n e m m involved in

learning and memory (Parker et al ... 1994 b). Inhibition of COX in rats redted in their

cognitive impakment and abnormal hïppocampat LTP (Bennett et al., 1992). And there

is some correspondence between the expression of mRNA of COX 1, COX II and COX

III and cell groups involved in AD, which may aüow for a widespread modest depression

of COX activity to produce focal CNS disease (Chan- et al., 1992 a and b). It

is known COX fdure would cause depression of ATP synthesis and bioenergetic

impairment, and such impairment resultiag h m primary ETC dysfimction has been

show to cause late onset and focal CNS disease (Wallace et al., 1988; Parker et al.,

1989 a and b, and 1990 b; Gato et al., 1990; Shapira et al., 11990; Shofïker et al., 1990

and 199 1; Howell et al., 1991). COX abnonnalities have also been linked to other

disorders characterizPd by cortical degeneration (Prick et al., 1983; Danks, 1983).

1.7 Ovtcupirssion of APP

In AD, the abnormal accumulation of PAP is thought to be due partially to

increased generation of APP and to abnormal processing of it (Selkoe, 1994 b and c). The

pathogenic mechanisms ofplaque formation include alterations in APP gene expression.

There is augmented expression of APP gene tnuiseripts in Dom's Syndrome and in

specinc aieas of the brain in AD (Higgans et al., 1988; Cohen et al., 1988; Neve et al,

1988; Johnson et al., 1990). Dupikation of the APP gene on chromosome 2 1 is sutFcient

for amyloid deposition and subsequent development of Alzheimer-type neuropatholow

(Hyman, 1992). Post-mitotic nemus overexpressing fûli-length APP showed

degeneration in vitro (Yoshikawa et al., 1992).

Marked increase in APP gene expression accompany environmental risk fàctors

for AD, such as brain trauma, ternporary ischemia and heat shock. The process is

potentiated by an ischemic Glu release that opens cellular CaZ' channels, inhibithg

glucose turnover and ATP production, and which is açcompanied by the generation of

(Roberts et al., 1991; Meier-Ruge and Bertoni-Freddari, 1997). It has been

postulateci that increased expression of the APP gene may alter the pn>cessiag of the

protein, produchg more insoluble PAP (Naibantogiu et al., 1997).

There is a relationship between APP overexpresion and abnomalities of COX

and mitochondrid structure (Shigenaga et al., 1994). The overexpression of APP via an

adenovinis vector in cultured normal human muscle fibres caused decreased COX

activity and structurai a b n o d t i e s of mitochoadria, in a dose- and durationdependent

manner. Decmsed COX activity preceded the rnitochondrial seuftural cbanges (Askanas

et al., 19%).

Oxidative stress is an important factor in the development of AD, as an

etiopathogenic oxidative mess hypothesis has been supported by severai experimentsl

findings (Olanow and Arendash, 1994; Beal, 1995; Hatman, 1995; Benzi and Moretti,

1995). Oxidative stress refers to the cytotoxic conse~uences of oxygen radicals - superoxide anion (.O;), hydioxy radical (-OH), and hydrogen peroxide (409 - which

are generated as byptuducts of normal and aberrant metabolic processes that utilize

molecular oxygen (Coyle and -ken, 1 993). The brain consumes a disproportionate

amount of the body's oxygen, deriving its energy almost exclusively fiom oxidative

metabolism of the mitochoadrial respiratory chah (Halliweli and Gutteridge, 1989).

ROS are continuously produced by mitochondria, and if the ceil's 4-oxidant defense

mechanisms are overwhehed, oxidative stiess occurs (Mecocci et al., 1998). Many

processes and enzymees in the brain also increase the levels of ROS and radical nitmgen

species, by producing them as by-products of theu teactions (Coyle and Puttfiken,

1993). Oxidative stress damages lipids, proteins and DNA, resulting in necrosis or

apoptosis (Sirnonian and Coyle, 19%).

Direct evidence supporthg increased oxidative stress in AD include (i) increased

brain Von, aluminum and mercury in AD capable of stimulating fkee radical generation,

(ii) increased lipid petr,xidation and deaeased polyimsatinued fatty acids (PUFA) in AD

brain, and increased 4hydroxynonenal (HNE), an aldehyde pmduct of lipid peroxidation

in AD ventricular fluid, (iii) increased protein and DNA oxidation in AD brain, (iv)

diminished energy metabolisni and decreased COX in brain in AD, (v) AGEP,

malondialdehyde, carbonyîs, peroxynitrite, heme-oxygenase-l and superoxide dismutase

1 (SOD-1) in NFTs, and AGEP, heme oxygenase-1 and SOD-1 in senile plaques, (vi)

plus the faft tbat f5A.P can geaenite ROS. Indirect eviâence cornes hm many in viho

studies showing that f k r a d i d s are capable of mediatiag newon degeneration and

death, whether or not they are a primay or secondary (eg. to tissue damage) event

(Markesbery, 1997). It has been d e m o d that AD COX does act as a ROS generator

(Partridge et d, 1994; Lakis et al., 1995; Parker and Park., 1993, which would account

for the increased levels of Lipid peroxidation in AD brah and other indicators of

radical involvement (Subbaro et al., 1990; Volicer and Crino, 1990). There is a

relationship between the redox state of the ceii and the degeneration in AD (Smith et al.,

199 1 ; Behl et al., 1994 a). When the ETC is inhibitecl, electrons accumulate in the early

stages of the ETC (complex 1 and COQ), where they can be donated directly to molecdar

oxygen to give supemxide anion. This is detonficd by mitochondrial maaganese SOD

(MnSOD) and glutathione peroxidase. Chronic ROS exposure leads to oxidative damage

to mitochondrial and cellular pmteins, iipids, and nucleic acids, and acute ROS exposme

can inactivate the iron-suiphur centres of ETC complexes I, ïï, and III, and amnitase of

the Krebb's cycle, resulting in shutdown of mitochondrial energy production (Wallace

1997 and 1999). It is known PAP causes piasma membnme lipid peroxidation and

generation of ROS that coutribute to celi death ( M A C et al., 19%). The neu~otoxic

actions of PAP couid be blocked by antioxidants (Behl et al., lm), and relied on the

presence of reactive oxygen intermediates (Behl et al., 1994).

It hris been shown that PAP caused oxydical-mediated impahent of glucose

transport, Glu transport, and mitochondrial fünction in rat neocortical synaptosomes.

Several antioxidants prevented the PAP-induced impairment of glucose transport,

indicathg that Lipid peroxidation was causaiiy Linlred to the adverse action of BAP. It is

suggested that oxidative stress occUmng at syaapses may wntribute to the reduced

glucose uptake and synaptic degeneration that occurs in AD patients (Keller et al., 1997).

One mechanisrn ofoxidative stress is the nitmîion of tyrosine residues in proteins,

mediated by peroxynitcite breakdom The latter is a ceaction product of NO and

superoxide radicals. Oxidative stress is Iinked to NFT formaiion, and NO excitotoxicity

and expression is Iinked to the pathogenesis of AD ( G d et al., 1996).

Brain, and specifically neurons, are particularly vulnerable to impairments of

oxidative metabolism because of their tight dependence on wntinuous oxidation of

glucose to mainttain their structure and function. Several studies bave s h o w the btain

areas of greatest vulnerability in AD include those pacticularly sensitive to oxidative

irnpairments (BIass and Gibson, 1991). As weli, brain ceUs are at peat risk h m damage

caused by f k radicals. The brain has an extremely high rate of oxygen consumption,

and CNS nemonai membranes are high in PUFAs tbat are very vulwrable to Lipid

peroxidation @eh1 et al., 1994 a). The free radical attack on PUFAs causes lipid

peroxidation and organic peroxy radicals to buiid up in a seifperpetuathg cycle. As weii,

the abstracted lipid hydrogen atoms combine with the peroxy radicals to fom lipid

hydroperoxides which, in the presence of f m u s ions, decompose to alkoxy radids and

aidehydes. Thus, the end result is the generation of numemus toxic reactants that rigidQ

membranes by cross-linking, disrupt membrane integrity, and damage membraae proteins

(Coyle and Wârcken, 1993).

A critical teason why oxidative stress is important in AD, is that the aggregation

of PAP and other amyloidogenic APPdenved peptides depends on radical gemration

systems (metal catalyzed oxidation systems) which transfocm soluble peptides into

insoluble aggregating molecules. This involves radical-induced cross-linking of tyrosyl

hydroxyl groups of the P-amyloidogenic peptides, the primary event leading to amyloid

formation (Dyrks et al., 1992). The aggregation of PHFs, üke PAP fibrils, is ais0 linked

to incrpased oxidatîon. The hdings support the notion that the increase in redox

potential and an excess protein oxidation found in aghg brain provide a Link for the

aggregation of PAP fibres and PHFs (Schweers et al., 1995).

Of relevance to AD, is that energy failure due to anoxia or hypoglycemia caused

a marked efflux of Glu to achieve concentrations in the extracellular space compatible

with neurotoxic effects (Benveniste et al., 1984; Katchman and Hershkowitz, 1993).

Activation of glutamate-gated cation channels is the most important source of oxidative

stress in the brain. These two mechanistus can act in concert to produce

neurodegeneration (Coyle and hinfatcken, 1993). In hua, active glutamate refeptors

(NMDA and ~aminoo3-hydroxy-5-methyl4i~~1epmpio~-bte) cause a

depletion of ATP and the induction of lactic acidemia (Retz and Coyle, 1982). The acidic

conditions favor the liberation of cellular FeZ' which promotes the Fenton reaction and

the Liberaiion of H202 (Hailiwell and Gutteridge, 1989). Related to thiq it is known

NMDA receptor stimulation produces marked elevations in -0; and -OH (LafonCazal

et al., 19 ). As well, NMDA receptor-mediated stimulation of phospholipase A, and the

subsequent release of arachidonic acid (AA) leads to the generation of oxygen radicals

(Dumuis et ai-, 1988; Lazararewicz et al., 1988). Added to this, AA and oxygen raâicals

enhance the release of Glu a d inhibits its uptake inactivation by neurunal and glial

transporter processes, promoting a selfperpetuathg cycle (Peliegrini-Giampietro et aï.,

1988; Williams et aï., 1989).

Accumulating evidence links apoptotic pathways to neurodegeneraîion in AD.

Histochemical studies report apoptosis to be a pathological feature of AD (Su et al.,

1994; Cotman and Anderson, 1995; Lassmann et aï., 1 995; Smale et aï., 1995). The loss

of hippocampal neurons by apoptotic ceU death is a prominent feature of AD (Smale et

al., 1995; Cotman and Su, 19%; Li et al., 1997; Su et al., 1997). One potential factor

contributhg to the susceptibility of these cells to premature death arises h m the

cytotoxic effects of PAP deposition at or nea. sites of neuronal degeneration Cultureci

human cells, including nemns, undergo apoptosis when treated with PAPs (Loo et al.,

1993; LaFeria et ai., 1995; Forloai et aï., 19%), and n e m m undergohg apoptosis

generate elevated levels of cytotoxic PAP species (LeBlanc, 1995; ûervais et al., 1 999).

This agrees with the proposition that the elevated PAP that occurs due to genetic

predisposition or other physiological factors, pmvokes partial activation of apoptosis in

susceptible neurons. These sensitized cells then produce more PAP, leading to a self

perpetuathg cycle which culminstes in the progressive neuronal loss seen in AD

(Gervais et al., 1999)

As weil, caspases, the primary mediators of apoptosis have been shown to be

involved in proteolytic cleavage of APP and in the biogenesis of arnyloidogenic PAP

species. Caspase-3 was found to be elevated in dying neurons of human AD brain. It was

demonstrateci that caspizse-mediated proteolysis o c c d in hippocampai neurons in vivo

during acute brain injury (ceil death). The caspase-cleaved APP also co-localized with

senile plaques and increased the rate of PAP formation in neuronal celis (Gervais et al.,

1999). It was also s h o w that there was enhanced PAP formation in celis harborhg the

Swedish mutation of APP, and that this was dependent on caspase proteolysis at the P-

secretase site (Gwais et al., 1999).

It is pmposed that cleavage of APP at endogenous caspase consensus sites

compts the nomial, non-amyloidogenic intraceliular pmssing of APP. The following

steps may then lead to a eifperpetuating cycle: caspase-3-mediated tmcaîion of AFP

at the COOH temiinus, adulteration of normal APP pfocessing with shunting of the

residual polypeptide toward an amyloidogenïc pathway, generatïon of elevated levels of

PAP resuItiag in PAP-induced neuronal stress, progressive upreguiation a n h activation

of caspases, and exacerbiited APP proteolysis (Gervais et al., 1999). Thus, APP is a

target for fertain caqmxs and is involved in apoptotic pathways (Grnais et al., 1999;

Weidemann et al., 1999). Elevated APP levels have been repozted in dying neurons

(pames et ai., l998), which could potentially feed more APP into the cycle (Weidemann

et al., 1 999).

It has recently been shown that mitocbondria wntain a caspase-Wre enymatic

activity in addition to three biological activities previously suggested to participate in the

apoptotic process: (a) cyfochrome c; (b) an apoptosis-induchg fnctor (NF) which causes

isolated nuclei to undergo apoptosis in vitro; and (c) a DNAse activity- Al1 of these

factors are released upon opening of the PTP. Experimental data suggest that caspase-2

and -9 zymogens are essentialiy localized in mitochondiia and that the dismption of the

outer mitochondrial membrane occUmag early during apoptosis may be critical for theü

sub-cellular redistribution and activation (Susin et ai., 1999). Loss of mitochondrial

barrier fünction during newonai damage h m ischemia or other insults (e-g. AD

pathology) therefore may play an important d e in making certain caspases available to

participate in apoptosis (Krajewski et al., 1999). In support of caspases and apoptosis

king involved in AD, the protein levels of both caspase-2 and -3 were significantly

increased in AD brains (S himohama et ai., 1999).

Of consequence to AD, neurons are particuiarly Milnerable to degeneration by

apoptosis. inducers of apoptosis (e.g. PAP, oxidaîive damage, low energy metaboüsm)

are also present in AD braias. Some nemm in vulnerable regions of AD brain show

evidence of DNA damage, nuclear apoptotic bodies, chromatin condensation, and the

induction of select genes characteristic of apoptosis in celi culture and animal models

(Cotman and Su, 1996). Evidence supporthg the involvement of apoptotic pn>cesses in

nedegeneration in vivo include the hding thaî apoptotic mediaîor proteins lüce Par4

are increased in newons of AD braias (Guo et d, 1998). Expression of mutant APP

carrying FADmutations haveinducedapoptosïs, ïnâicirtingtbatspoptosismaycontribute

to neuronaI loss in FAD CyamafSuji et al-, 19% a and b; Zhao et al., 1997). This bas been

shown to be dependent on a short stretch witbin the cytosolic domai. of APP, and is a

site where caspase cleavage occurs (Wiedemann et al., 1999). Caspases may thus play

an important rule in the generafion of PAP as well as in the d a death of neurons by

apoptosis in AD (Gervais et al., 1999).

Mitochondrial dysfhction appears to occur in AD (Beal, 1992), and

mitochondria provide a major switch in the initiation of apoptosis. A de- in

mitochondrial membrane potential (A*) causes opening of the mitochondrïal inner

membrane channel, the PTP, which has bew implicated as a critical e f f i r of apoptosis

in a variety of non-neural ceils (Zoratti and Szabo, 1995; Petit et al., 1996; Green and

Reed, 1998; Tatton and Chalmers-Redman, 1998). Opening of the h4TP causes collapse

of the proton motive force (A*), sweliïng of the miiochondrial inner membrane, and

release of death-promoting factors, such as cytochrome c, AIF, and latent foms of

caspases (Wallace, 1999). Opening of the PTP and the accompanying death of the ceil

can be initiated by the mitochondrion's excessive uptake of Ca2+, increased exposure to

ROS, or decline in energetic capacity (Zoratti and Szabo, 1995; Liu et al., 1996;

Bnistovetsky and KLUigenberg, 1996;Green and Reed, 1998; Mano et al., 1998; Susin

et al., 1999; Eanishaw et al., 1999). Thus, a marked reduction in mitochondriai energy

production and a chmnic increase in oxidative stress, as known to occur in AD, wuld

theoretically activate the PTP and initiate apoptosis.(Waliace, 1999).

Oxidative stress and apoptosis are closely ü n k d It is known that H202 induces

apoptosis in different cells, uicluding rat newons (Whittemore et al., 1994), and human

neuroblastoma cells (Zhang et al., 1997). PAP can induce both necrotic (Behl et al., 1994

b) and apoptotic cell death, which can also be caused by oxidative damage (Lw et ai.,

1993 ; Hockenbery et al., 1 993; ForIoni et al., 1993; Watt et ai., 1994;Wttemote et ai-,

1994; Anderson et ol, 1995; LaFerlaet d, 1995). BAP-induced wurotoxicity correIates

with elevated H202. A large number of antioxidants and fke radical scavengers protected

h m PAP toxicity and inhibited peroxide accumulation. PAP increased lipid

peroxidation that could be iabibited by antioxidants @eh1 et al., 19!92 and 1994 a).

Zhang et a2 (1997) demonstrated that H202-induced apoptosis altered APP processiag,

producing an apoptosis-associated 5.5 kDa APP COOH-terminal m e n t , suggesting

PAP is a mediator of apoptosis. The generation of H202 by PAP reduces the flow of

electrons through the ETC. As well, H,O, is converted to the -OH-, which intedixes with

the ETC directly and via lipid peroxiùation of the mitochoadriai membrane (Behl et al.,

1994 a). The increased generafion of ROS has also been düectly linked to neuronal

apoptosis in fetal neurons of Down's synàrome (l3usciglio and Yankner, 1995). Zhang

et al. (1997) postdate that a cycle may exist between Ha, PAP and apoptosis. PAP

induces increased H202 levels, which enhances the amyloidogenic, and inhibits the

normal a-secretase, processing of APP, pducing more PAP. As well, increased H202

and P A P levels induce apoptosis, which stimulates more PAP production.

To date, deficits in energy metabolism and COX activity, oxidative stress and

lipid peroxidation of biologicai membranes, overexpression of APP, amyloidogenic

processing of APP and the accompanying excessive production of BAP species, as weil

as neuronai degeneration and apoptosis, have ai i been irnplicated in the pathogenesis of

AD. However, the exact mechanisms underiying the pathophysi010gy of AD have yet to

be elucidated. Our initial aim was to identw APP effeftor binding proteins in order to

deduce a mechanism whereby APP and its amyloidogenic derivatives may be involved

in the pathogenesis of AD. Through the use of a yeast two-hybrid system, it was

discovertxi that APP interacted with COX 1, a catalytic subunit of Complex N of the

mitochondnal ETC. This hding may, therefore, link APP (and pRhaps its

amyloidogenic derivatives), to the deficits in energy metabolism (and spcincally

reduced COX activity), that have been weii documentecl in AD. From this hding, our

hypothesis was that APP is targeted to, and resides in, mitochondria, wbere, uader some

circumstances (e-g. ovecexpression of APP), the duect and aberrant involvement of APP

and its potentidy amyloidogenic derivatives with COX 1 causes (i) deficits in COX

activity (bioenergetic defat), (ii) generation of ROS and its accompanying lipid

proXidation, (Si) abernint amyloidogenic pmxssing of NP, and 0 subsequent

wmnal apoptosis. The pathogenic postulates (i) to (iv) have alrrady been demonstrated

to occur in AD. However, we have tried to tie these aspects together into a working

model, as our hypothesis states.

To substantiate our hypothesis, we wished to fairy out the foilowing experiments:

1, Dctermine what regions of APP are involved in the interaction, by restriction

enzyme (REFbased deletions.

2. Demonstrate tbat overexpression of APP in nemb1astoma ceils causes a

selective deficit in COX activity, increased generation of ROS and lipid

perorùdation, and incteased apoptosis.

3. Show that APP is targeted to, d Iocalizcd in, mitochotxiria

4. Demonstrate that PAP also resides in mitochondria, suggesting APP is piocessed

in this organelle.

3.0 MATERIALS AND METHODS

3.1.0 Pnparation of Pbrnid/APP DNA Comtn~cts

3.1.1 pAS2-1IAPP- comtnrct

APP, cDNA containhg 3432 base pairs (bps) plus 15 bps of the 5' untranslateci

region (üTR) was received inserted into the pGEM9zfplasmid (Promega) (Figure 2). To

ampli@ the pGEM9zVAPPT10B432 constnict, 100 pl E. coli XL1-Blue supercompetent

cells (Stratagene) were transformeci with 1 pg ~ G E M ~ z ~ ~ A P P - ~ ~ , followiag the

standard protocol as per Promega. That is, the bacteria/DNA mixture was cold shocked

on ice 0.5 hour (hr), foilowed by heat shock for 1 minute (min.) at 42%, and then put on

ice for another 2 min. Next, 1 mililitre (ml) Luria-Bertani (LB) broth was added and the

Figure 2 Schematic of pGEM9zfrestriction map and MCS. The APPm32 cDNA was inserted between the EcoR 1 a d Sa1 1 RE sites in the MCS of the pGEM9zfvector. The sequence shown ~ m q O f . I d s to RNA synthesized by SP6 RNA polymerase and is wmplementaq to RNA synthesized by T7 RNA polymerslse. Some SeQuence reference points: SP6 RNA polymerase ttaascription initiation siîe, (1); T7 RNA polyniera~e transcription initiation site, (55); SP6 RNA polymerase promoter, (2909-6); T7 RNA polymerase promoter, (50-72); multiple clonhg sites, (5-54), lac2 stait d o n , (107); beta-lactamase (Ampr) coding region, (1264-2 124). (Adapted h m hmega).

mixture incubated for 1 hr at 37°C on a rotating incubator (250 revolutions per minute

[rpm]). Ampicillin (amp) (LOO pg/rnl - this is the concentration used in al1 fùture

references to ampiciliin, uniess otherwise specified) was added and the E.

coli/pGEMWAPPm,,/amp mixture was incubated overnight at 3TC and 250 rpm.

About 300 pl of the incubate was added to 1 litre (L) LB/amp broth, and the mixture

incubated ovemight at 3TC and 250 rpm. A plasmid maxiprep (QIAGEN's P I m i d

Maxi Kit) was perfomed to purify and exhact the flEM9zfAPP-32 D m &se

concentration was determineci photometrïdy h m the equation:

[DNA] pg/pl= A x 50 (for double stranded DNA) x (1000 [pl

water in cuvette] + pl sample DNA) / (1 000 x pl sample DNA).

To v e m the DNA isalated by the plasmid prep was the correct plasmid

constnict, the DNA was restricted with with Bgl II. Reaction mixture: 20 pl distilleci.

deionized water (ddwater), 2 pl buffer, 2 pg ~GEM~z~ /APP-~~ , 2 pl Bgl11. This was

incubated in a water bath for 3 hr at 3TC. following which, a 0.8% agarose gel electro-

phoresis (AGE) of the Bgl II-restncted pGEMWAPPm32 was pe&omed.

To prepare the APP, fiagrnent for insert into pGEMSzf plasmid (Promega)

(Figure 3), the pGEM9dAPPm, comtruct was restrïcted with Spe 1. Reaction

mixîure: In each of five tubes, 40 pl ddwater, 5 pl buffèr, 5 pg pGEM9zVAPPm32, 2

pl Spe 1. The mixtures were incubated for 3 hr at 37T, followed by a 0.8% AGE. The

appropriate APPmm, DNA was cut h m the gel, and the DNA isolated and purified via

QIAGEN's QUEX lï Gel Exhacrion Kïî.

To prepare the pGEMSzf plasmid to receive the APP,, insert, the plasmid

Figure 3 Schemaîic ofpGEMSzfrestriction map and MCS. The pGEMSzfplasmid was restricted with Spe 1, and the Spe 1 -testricteci APP, was ügated into this site, to the constnict pGEMSzDAPPmm. Some sequence refetence points: TI RNA polymerase tnuiscription initiation site, (1); SP6 RNA polymerase transcription initiation site, (126); multiple cloning sites, (10-1 13); lac2 start d o n , (165); lac operon sequences, (28242984,lS 1 -3 80); lac operator, (1 5 1-20 1); beta-Irictamase (Ampr) ooding region, (1 322-2 1 82).(Adapted fkom Pmmega).

was fkt ampli6:ed by transfonning 100 pl E. coli XL 1 -Blue supercompetent ceils with

1 pg pGEM5zf DNA, protoc01 as above. A plasrnid maxi- (QIAGEN's Phmiid

Mmi K . ) was performed and the plasmid coostriLct verified by RE analysis. The

pGEMSzf plasmid was then restricted with Spe 1 to prepare it îo accept the Spe 1-

resîricted APPm32 fragment. Reaction mixture: In five separate tubes, 70 (11 ddwater,

10 pl buffer, 10 pg @EMS&, 3 pl Spe 1. The mixhires were incubated ovemight at

3TC, and then subjected to 0.8% AGE. The Spe 1 - d c t d pGEM5zfbad w a ~ CU^

h m the gel, isolated and purifIed via QIAGEN's QUEXI1 Gel fifraction Kit, and its

concentration determineci photometrically.

To prevent the plasmid h m self-ligating before accepting the insert, the Spe 1-

restricted pGEMSzf was de-phosphoryiated, as per Promega, as follows. Fkt , the

amount of picomole @mol) of ends of Linear double stranded DNA was calculateci,

according to the formula: (pg DNAlKb size of D M ) X 3.04 = pmol of ends. Reaction

mixture: 10 pl calf intesfinal alkaline phosphatase ( C M ) 1OX d o n buffer, 0.03 u

CIAP/prnol of ends of DNA (Le. 0.3 u CIAP per 10 pg of pGEMSzf), 20 pg pOEMSzf,

ddwater to make a £inal volume of 100 pl. The miction mixture was incubated at 3 7 T

for 30 min, after which, an equal volume of CIAP was added and the mixture incubated

for another 30 min at 3TC. The reaction was then stopped by adding 2.0 pl of 0.5 M

EDTA and heated at 6S°C for 20 min. The de-phosphoylated, Spe 1 - d c t e d pGEMSzf

was precipitated and p d e d with phenol/chlomform/isoamyl alwhol (PCIAA) and

ethanol, as per Pmmega, as foilows: An equai volume PCIAA was added to the DNA

mixture, vortexed for 1 mia, and centrifuged at 12,000 rpm for 5 min. The upper

aqueous layer was transfèrred to a new tube, to which was added 0.1% 3 M sodium

acetate (pH 5.2) and two volumes of 1Wh ethanol, followed by storage at -20% for 30

min, and then cenMùgatïon at 12,000 rpm for 30 min. The supematant was discarded

and 200 pl 70% ethanol a d d d This was centrifuged for 15 min at 12,000 rpm. The

supematant was discarded a d the pellet aiIowed to dry, after which it was re-suspended

in 50 pl ddwater.

The M'Pm, hgment was ligeted into the de-phosphorykd, Spe 1 -restncted

pGEMSzf, using Clontech's Ligutioa &press Kit, to create the construct,

pGEMSzEIAPPTIwJa. Next, 100 pl E. coli XLlBlue supercompetent ceUs were

transformed with the whole ligation mixture, as per Pmmega. Followhg this, 100 pl of

re-suspendeci transformed cells were spread onto an LB/ampTisopropylthiogaiactoside

pTG]/Xgal agar plate (as per Sambmk et al., 1989), and incubated ovemight at 37%.

Ifthe APP DNA was Uiserted into the plasmid, it would dimpt the LocZgene, resulting

in white colonies. To determine the correct orientation of the insert, ten white colonies

were selected and each colony was added to separate tubes of 3 ml LB/amp. These were

incubated ovemight at 3PC and 250 rpm. M k 2 ml of euh incubate were pelleted

(5000 rpm for 10 min), a plasmid rniniprep (QIAGEN's Plamid Mini Kit) was

perfomed. Each plasmid prep DNA was restricted separately with Sac 1 and Pst 1.

Reaction mixture: 20 pl ddwater, 3 pl bbuffer, 1 pg DNA, 1 pl Sac 1 or Pst 1. The

reaction mixtures were incubakd for 2 hr at 3TC, after which, a 0.8% AGE was

performed. The contents of the tube h m which the correctly onented E.

coli/pGEM5zfïAPPm, construct origïnated were added to 1 L LB/amp and incubated

ovemight at 37°C and 250 rpm. This was followed by a plasmid maxiprep (QIAGEN's

PIasmid Mmi Kit) to isolate and puri@ the pGEMSzf7APP-, construct,

To prepare the AFPma hsert end the GAL-4 bindhg domain (BD) plasmi4

pAS2-1 Figure 4), to receive the insert, both the pGEM5zVAPPma3 construct and

pAS2-1, which bad been previously amplified and purifIed as above, were

simultaneously restncted with Nco 1 and Sa1 1. Reaction mixture: Five separate tubes of

50 pl ddwater, 10 p1 buffkr, 10 pg DNA, 3 pi Nco 1 and 3 pl Ml . The reaction was

incubated overnight at 37°C foilowed by 0.8% AGE.

The appropriate DNA bands were cut h m the gel and the DNA eluted into TAE

buffer by standard membraae electrophoresis procedures. The DNA was purified and

precipitated fiom the d t i n g TAFdDNA mixture by PCIAA and ethanol, as per

Ehmega The APPma was then Ligated into the Nw l/Sall -restricted pAS2- 1, using

Clontech's "Ligation Express Kit". Following this, the whole ligation mixture was added

to 100 pl E. coli DHSa ceus, and the ceils transformed as per Promega. The E.

~ol i /pAS2-1/APP, ,~~ pellet was re-suspended in 100 pl LB broth, spread on to an

LB/arnp plate and incubated overnight at 3TC. Ten colonies wen added to sepiirate tubes

of 5 ml LB/amp and incubated overnight at 3TC and 250 rpm. A plasmid miniprep

(QIAGEN's Plusrnid Mini Kit) was performed on each incubate.

To ver@ the correct wnstruct was present, the pAS2-I/APf was restricted

in separate tubes with Pst 1, Bgl II or Hiad m. Reaction mixture: 25 pl ddwater, 4 pl

buffer, 2 pg DNA, 2 pl RE. The mixnaes were incubated for 2 hr at 37%, and then

subjected to 0.8% AGE.

ACT 6TA f ~ 6 CC6 6TA T ~ G C U TAC CCA 6CT ïT6 ACT

61 CGA $1 M A 6CC MG CTA A n CC6 6/c GM ïïï Sali Psi StoF

Figure 4 Schematic of pAS2-1 restriction map and MCS. (Unique restriction sites are in bold). Location of Feaîures: 2p origin of replication, (1-1348); TRPl coding sequences; Start codon (ATG), (18841886); Stop codon, (2256-2258); CYH2 gene, (3765-4627); Promoter, Fragment h m &cchmomyces cerevisiae contaïning the ADH 1 promoter, (4767-5474); GALA bindiog domain polypeptide; Start codon (ATG), (5502-5504); GAL4 codons 1 - 147, (5502-5944); MCS, (5970-60 1 5); Transcription temiination sigoal; Fragment carrying the S. cerevisiae ADH 1 tenniriator, (603 1-6223); Translation stop codon, (60636056); Ampiciilin mistance gene, (7403-8263). (Adapted h m Clontech).

The contents of wmxt incubates were added to 1 L LB/amp broth and incubated

ovemight at 3TC and 250 rpm. A plasmid maxiprep (QIAGEN's Plasmid M a i Kit) was

perfomed to isolate and puri@ the pAS2- l/APPma wnstruct Figurp 5 shows a

schematic of the APP-, insert-

3.12. P A S ~ - ~ I A P P ~ ~ COOS~~PC~

To isolate the APP-- fragment, pAS2-l- APP mm was restricied with

EcoR 1 and subjected to 0.8% AGE. Reaction rnix: Five separate tubes of 40 pl ddwater,

5 pl bunei, 1 O pg pAS2-1,3 pl EcoR 1. The mixtures were incubated ovemight at 3TC.

The pAS2- 1 and APP,,, bands were cut out of the gel, and the DNA extracted with

QIAGEN9s QZEXII Ge2E;lctraciion Kit. To ver@ the APP7MIN7MIN12020 fhgment was present,

an additional 0.8% AGE was perConned on the hgment alone (data not shown).

To prepare pAS2-1 to accept the fragment, previously ampLified and

verified pAS2-I plasrnid was restricted with EcoR 1. Reaction mixtrne: Five separate

tubes of 40 pl ddwater, 5 pl buffer, 8 pg pAS2-1,2 pl EcoR 1. Murhaes were incubated

overnight at 3TC, foliowed by 0.8% AGE and extraction of the DNA h m the gel with

QIAGEN's QZEXIT Gel Extraction Kit.

To prevent self-ligation, the EcoR 1-restricted pAS2-1 plasmid was de-

phosphorylated according to Promega, except thaî five tubes were used, each with about

25 pg EcoR 1-restricted pAS2-1,O.l u C M , 30 pl CiAP Buffer, 270 pl ddwaîer. The

de-phosphorylated, EcoR 1 -restricted pAS2-1 was purified and precipitated with P C M

and ethanol, as per Promega, and the concentration of the de-phosphorylated DNA was

Membrane

Figure 5 Schematic ofhuman APP,, construct. The APP-, coostnrt consisted of nucleotides 1-2254, plus a 15 base pair 5' UTR and a 329 base pair 3' UTR The constmct included most of the wding region of NP,.

photometricaily determined.

The Ammnrn U e n t insert was ligated into the EcoR 1-restricted and de-

phosphorylaîed pAS2-l, using Boebringer-Mannheim's R@d DNA Ligution Kif, with

foliowing modifications: 50 ng pAS2-1,500 ng APPmmo, 30 pl dilution buffer, 30

pl ügase buffet, 3 pl T4 DNA ügase, and the ligation reaction was incubeted at m m

temperature for 30 min- Next, 100 pl E. coli DHSa cells were M o r m e d with the

whole ligation mixhue following Promega'sprotr>col, except that the E coli/plasmidmix

was incuhed ovemï@ at 3TC. The ovemight incubate was then spread ont0 four

LB/amp plates which were incuôated ovemight at 3TC.

To check for correct ligation, five colonies were selected and each put into

separate tubes of 3 ml LBlamp broth. They were incubated for 48 hr at 3TC and 250

rpm. A plasmid miniprep as per Pmmega's Wizurd Plasnid MiniPrep Kil was petfiormed

on the pAS2-l/APPnwnm incubaies, and the concentration determined

photometrically. To ver* correct ligation and orientation, the pAS2-l/APPtn/N-n020

DNA h m each colony was restricted with Sac 1. Reaction mixture: 35 pl ddwater, 3 pl

buffer, 1 pg DNA, 1 pl Sac 1. The reaction was iacubated ovemight at 37C, and then

subjected to 0.8% AGE.

An appropriate vial containing the correctly oriented APP insert was transferred

to 1 L LB/amp broth and incubated ovemight at 3TC & 250 rpm. A plasmid maxiprep

(Promega Wizord PIaPmid M i PIep) of the amplined pAS2-l/APP-- wostnrt

was performed. To re-verQ the correct construct, the pAS2-I/APP-,, preperation

was restricted with Sac 1. Reacton miu: 35 pl ddwater, 3 pl buffery 1 pg DNA, 1 pl Sac

1. The reaction was incubated for 2.5 hr at 3 X , after which a 0.8% AGE was performed.

3-12 p A S 2 - 1 / A P P ~ ~d pAS2-I/APP-- ~ ~ ~ ~ t r u c t s

To create fkther deletions of APP, the foiiowing REs were used:

(i) Barn Hl. Reaction mixture: Five seperate tubes of 40 pl ddwater, 6 pl buffer, 8 pg

DNA, 4 p1 BamH 1, incubated ovemight at 3 T C . (ii) Pst 1. Reaction mixture: Five tubes

of 45 pl ddwater, 5 pi buffer, 10 pg DNA, 3 pl Pst 1, Urubated for 3 ht at 3TC. A 0.8%

AGE of BamH 1- and Pst 1-restricted pAS2-IIAPP-,, was perfomed.

The appropriate gel slices containhg the pAS2-l/APPm-T,, and pAS2-

l/APPm-no8 DNA were cut h m the gel, and the DNA extracted by membrane

electrophoresis. The pAS2- l/APPm-Tlw and pAS2- 1 IAPP-- fhgments were

purified and precipitated by PCIAA and ethanol, as per Promega, and the concentration

determined photometrically. This was foliowed by ligation of the EcoR 1 -restricted

pAS2- 1 /APPnm-nm, B a d 1 -resnicted pAS2- 1 /APPmT1, and Pst 1 -resûicted

pAS2-l/APPm770ME,, constructs with Boehringer-Mannheim's Rqid DNA Ligation Kit,

with the following madincations: 30 pi DNA dilution buffet, 700 ng DNA, 30 pl ügase

buffer, 3 pl T4 DNA ligase, and the reaction mixtures were incubateci for 1 hr at m m

temperature. Next, tfvee tubes of 100 pl E. coli DHSa cells were transformeci with the

whole ligation mixtures, uskg the pmtocol as per Promega, except for the foiiowing

modifications: Mer adding the DNA to cells, they were stored on ice for 30 min.,

subjected to 42% for 1 min., and put back on ice for 2 min. The cells were then

transferred to 3 ml LB broth and incubated for 2 hr at 37% and 225 rpm. 2 ml of the

incubates were removed and pelleted (centrifugecl at 5000 g for 5 min), followed by

resuspension in 100 pl LB broth. This was spread on LB/amp plaies and incubated

ovemight at 37'C. Two colonies nOm eaçh of the three LB/amp plates (tramdomid with

ligated pAS2- 1 /APP-mo, pAS2- 1 /APPmT,, and pAS2-1 /APP-& were

selected and put in six separa& vials of 5 ml LB broth. The tubes were incubated for 1

hr at 3WC and 250 rpm and then had 10 pl ampicillin added They were incubated

overnight at 30°C and 250 rpm, atter which the incubates were added to 200 ml LB/amp

and iacubated ovemight at 3TC & 250 rpm. A plasmid maxiprep (Pmmega's Wkurd

MmiPrep DNA Pwzjïcation Systen) was peifonned on the incubates and the

concentration of the purïfïed DNA calculated photometrically.

The pAS2-1 /APP-- and PAS~-I/APP--~,, constrwts were verified ushg

Hind m. Reaction mixture: 22 pl ddwater, 3 pl buffer, 1 pg DN& 1 pl Hind m. The

teactions were incubated ovemight at 3TC, followed by 0.8% AGE.

3.1.4 pCMV/myc/mito/APP- c o a s t ~ c t

First, the pCMV/myc/mito plasmid and the pGEMSzVAPPm, wnstruct were

arnplified and verif7ed to be correct. To amplify pCMV/myc/mito plasmid, 50 pl of E.

coli DHSa (GibcoBRL) ceils were traosformed with 1 pg pCMVlmyc/mito plasmid

(Tnvitrogen). Protocol as per GibcoBRL, as foflows: 1 pg pCMVlmyc/rnito p l d d was

added to 50 pl DHSa competent cells, gently tapping the tube to mix. The cells were

incubated on ice for 30 min, then heat shocked at 37'C for 20 sec, and finally p 1 d on

ice for 2 min. Next, 1 ml of m m temperature LB was added and the mixture then

incubaîed for 1 hr at 3TC dé 250 rpm. A f k 1 hr, the incubate was centrifiiged at 14,000

rpm for 10 sec to peilet the celis. The pellet was re-suspendeci in 100 pl LB broth, and

spread on to an LBlamp plate. Three colonies of E. coli/pCMV/myc/mito and four

colonies of E. colÏ//pGEM5dAPPmt3 were selected to be added to separate tubes of

5 ml LB broth. These were incubated for 1 hr at 3TC and 250 rpm, at which time 10 pl

ampicillin (50 mg/ml) was added, and the incubertes were then incuhated ovemight at

3TC and 250 rpm. 3 ml each of the E. coIifpCMV/myclmito and L

coIi//pGEMSzVAPP-, incubates were t r a n s f d to 200 ml LB/amp (100 ug/ml)

broth, and incubated overnight at 3TC and 250 rpm. This was foilowed by plasmid

maxipreps of the incubates (Promegays Wùcrrd Plus Mmipep DNA Purijcation System),

with the concentration of the DNA king detennined photometrically. To v e w

plasmids, (i) pGEMSdAPP-, was reSfTicted with Sac 1, and (ü) pCMV/myc/mito

was restricted with Sa1 1. In both constructs, expected bands were seen on 0.8% AGE

(data not shown),

To isolate the -Pm, insert, the pGEM5zUAPPm, construct was restricted

with Spe 1 .. Reaction mumire: Two separate tubes of 50 pl ddwater, 8 pl buffer, 10 pg

DNA, 4 pl Spe 1. The reactions were incubated for 3 hr at 3TC, followed by 0.8% AGE.

The 2.62 Kb N P m m band was cut h m the gel, and the DNA extractecl and purifieci

with BioRad's PrepA-Gene DNA P1Pijcation System. The DNA concentration was

determined photometrically. The pBluescript II SK plasnid was then amplified by

transformiag 50 pl E. coli DH5a ceils with 1 pg pBluescript il SK plasmiâ, pmtocoI as

per GibcoBRL. Mer the traosformed celis had ken hcubating for 1 hr at 31%, 10 pl

ampicillin (50 mg/ml) were added and cells incubated overnight at 3TC. The 5 ml

incubate of E. colüpBluescript iI SK was added to 500 ml LBlarnp bmth, and incubated

ovemight at 3TC and 250 rpm. This was foilowed by a plasmid maxiprep of the E-

coliYpBluescript II SK incubate, using Pmmega's Wiiard plus MmiPreps DNA

Pwzf?cation System, afkr which, the DNA concentration was determined

photometridy.

The pB1ue-pt II SK was next restücted with Spe 1 to d o w it to accept the S p

1-reStncted APPm3, m e n t . Reaction mixture: Two tubes of 50 pl ddwater, 8 pl

buffer,lO pg DNA, 4 pl Spe 1. The madion was incubated overnight at 3TC, foiiowed

by 0.8% AGE of the Spe 1-iestricted pBluescript [I SK DNA The Spe 1-restricted

pBluescript II SK DNA band was excised h m the gel, and the DNA isolated and

purified via BioRad's PrepA-Gene DNA Pwifiution System. The DNA concentration

was determined photometricaily.

To prevent the pBluescript II SK vector h m self-ligating during the subsequent

ligation reaction, 14 pg Spe 1-restricted pBluescnpt iI SK was de-phosphorylated, as per

Promega. This amount was calcdated to contain 14.18 pmol ends, which at 0.03 u

CIAP/pmol end required a total of 0.43 u CIAP ( 0 . 5 ~ CIAP was used). The de-

phosphorylated, Spe l -restricted pBluescript 11 SK was purified using Bio Rad's PrepA-

Gene DNA Pu@cation Kit.

Next, to mate the pBluescript II SK/APPm, construct, 4 pg Spe 1-restncted

APPma was ligated into 1 pg de-phosphorylated, Spe 1-restricted p B l d p t II SK,

using B-M's Rapid DNA Ligaiion Kit. Pmtocol as in kit, except the reaction was

incubated for 15 min et m m tempetature. The whole ligation mixture was then added

to 100 pl E. coli DHSa celis, and the transformation protoc01 as per GibcoBRL was

followed The pmtocol is identical to that described for transformation of 50 pl E. coli

DHSa ceils, except the cells are k a t shocked for 45 secs. At the end of the procedure,

100 pl of re-suspended ceiis was spread onto LBIamp plates and incubated overnight at

3TC. This was followed by a plasmid miniprep to verify the correct orientation of the

insert, and subsequently a large p M d pnp of the appropriate pBluescript II

SK/APPm, construct (data not shown).

To prepare the pCMV/myc/mito plasmid to accept the APPma insert, it was

simultaneously restricted with Sa1 1 & Not 1 REs. Reaction mixture: Two tubes of 50 pl

ddwater, 10 pl buffet, 10 pg DNA, 3 ~1 each enzyme. The reactions were incubated

ovemight at 37"C, &et wbich a 0.8% AGE was @orme& The 4.9 Kb Sai lMot 1-

resûicted pCMVlmyc/mito band was cut h m the gel and the DNA isolated and p d e d

with BioRad's Prep-A-Gene DNA Purifcation System. The DNA concentration was

detentljlled photometridy.

The next step involveci removal of the NP,, W e n t h m the pBluescript

II SWAPP-, tmnscript, to aUow the fiagment to be Ligated into the pCMV/myc/mito

plasmid. To achieve this, the pBluescript 11 SK/APPmm construct was restricted

simdtaneously with Sa1 1 and Not 1, the APPma DNA was isolated and purified h m

an agarose gel, md subsequently iigated into Sa1 lNot 1 - d c t e d pCMV/myc/mito,

using Boehringer-Mannheim's RupidDNA Ligation Kit. The whole ligation mixture was

added to 100 pl E. coli DHSa cells, and the transformation pmtocol as per GibcoBRL

was followed. At the end of the transformation pmcediue, 100 pl of re-suspeisdad ceiis

were spread ont0 LB/mp plates and incubated ovemight at 3TC. This was foilowed by

a plasmid mùiiprep and RE analysis to venfy the correct comtmct, and a plasmid

maxiprep of appropriate prepaiation to isolate and puri@ the pCMV/myc/mito/APPmm

wnstruct (data not shown).

3-13 PEGFP-N~IAPP-~~ ~as&rict

In summary, tbis involved cutting the APP,,, inseit out of a

~ G E M ~ z ~ Y A P P ~ ~ ~ wnstnict with EcoR 1, and ügating it into an EeoR 1-restricted

pEGFP-N 1 plasmid. The APPmsflm cDNA was received identically inserted into

pGEM9zfa~ W ~ S APPm,3432.

T0 PrePare the AwmïW-T~n, *et (ApPms9 bases 1-1795),

pGEM9&7APP,,, was restricted with EcoR 1. Reaction mixture: Two tubes of 40 pi

ddwater, 5 )i buffer, 12 pg DNA, 3 pl EcoR 1. The reaction was incubated ovemight at

3TC, and was followed by0.894 AGE.. The 1.8 Kb band (APPaNT,m) was cut h m the

gel and the DNA extraçted via membrane electmphoresis. The APP,,,,,, IlNA

£kgment was purified via PCIAA and ethanol as per hrnega, and the concentration

determined photometridy.

pEGFP-NI was ampüned by ttansformuig 100 pl E coli DHSa ceiis with 1 pg

pEGFP-N 1 plasrnid, and foltowing the transformation protocol as per Gibco BRL. AAer

the transformation p r d u r e , 50 pl of the 100 pl re-suspended ceU pellet was s p d on

LB/kaaamycin (50 &mi) m y c i n @an) concentration used in aii procedures was 50

pg/rnl] plates and incubated ovemight at 3TC. Sevaal colonies were selected and ali

added to 1 ml L B h bmth, which was incubated overnight at 3TC and 225 rpm. The

iccubate was traasfened to 500 ml L B b and Uicubated ovemight at 3TC and 225 rpm.

A plasmid maxiprep (QIAGEN's Plamid M i Kit) was per6iomied and the

concentration detemUeed p h ~ t o r n ~ c a i i y . To v e w that it was pEGFP-NI isolated, the

plasmid prep was restricted with EcoR 1. Reaction mixture: 20 pl ddwaîer, 2 pl buffer,

1 pgDNA, 1 ~EcoR1.ThedonwasincubatedforlS hrat3TC,afterwhichitwas

subjected to a srnail 0.8% AGE. To re-ver* pEGFP-NI, two more restrictions of

pEGFP-NI with EcoR 1 were carried out, followed by 0.8% AGE. The last restriction

showed only the 4.7 Kb band (data not shown) Confident that the correct plasmid was

present, the 4.7 Kb pEGFP-NI band was cut h m the gel and the DNA extracted via

membrane electrophoresis. The isolated DNA was &en pudïed via PCIAA and ethawl,

as per Promega, following which the concentration was detennined photometridy.

To prevent the EcoR 1-restricted pEGFP-NI DNA h m self ligating in the

subsequent ligation steps, it was de-phosphorylated, as per Promega Y was calculated

(Promega) that 15 pg EcoR 1 -restricted pEGFP-NI containeci 1 O pmol ends. At 0.03 u

CIAP per pmol ends of DNA, 0.3 u CIAP was used in the tigation miction.

mixture: Four tubes of 15 pg EcoR 1-restricted pEGFP-NI, 13 pl ddwater, 5 pI 10X

CIAP buffer, 0.3 u CIAP. The reaction protocol as pet Promega was foliowed. The de-

phosphorylated, EcoR l -restricted pEGFP-NI was precipitated and p d e d via PCIAA

and ethanol as per Promega, with the concentration king determined photometncaily.

The next step involved Ligation ofAPP,,, into the de-phosphorylatad, EcoR

I -restricted pEGFP-NI. As calculaîed (Pmmega), a 1 :3 ratio of vector to insert was

equivalent to 1 pg pEGFP-NI and 1.1 pg APPmTIm. For the Ligation, Boehringer-

Mannheim's Ropid DNA Ligcrtôn Kit was used. The kit protoc01 was followed, except

for the following parameters: 1.12 pg APP,,,,, 1 .O8 pg pEGFP-NI , 3 pl 1 X dilution

buffer, 13 pl Ligase buffet, 1 -4 pl T4 DNA ligase, and the rieaction was incubated at room

temperature for 1.5 hr.

To ampli@ the construct, 100 pf E. c d DH5a ceiis were transforrned with the

whole Ligation mixture, and then the protocol as per G i b BRL was followed. The

transformed celis were pelletai (14,000 rpm for 20 sec) and re-suspended in 100 pl LB

broth, which was subsequently spread on an L B h n plate and incubated ovemight at

3TC.

To verie that the ligation was successfid, ten colonies were selected and added

to ten separate tubes of 4 ml L B / ' bmth. These were incubated overnight at 3TC and

225 rpm, following which, a plasmid miniprep (QIAGEN's PIasmîd Minî Kif) was

performed on each incubate. Having calculateci the concentration of the DNA

photometridy, 3 pg DNA h m each tube was testricted with EcoR 1. Reaction

mixture: 40 pl ddwater, 6 pl buffer, 3 pg DNA, 3 pl EcoR 1. The reactions were

incubated for 4 hr at 3TC, followed by 0.8% AGE.

To v- that the insert was correctiy oriented, the remaining contents of the tube

with the ligated P E G F P - N I / A P P ~ ~ - ~ ~ ~ constructs were dded to 300 ml L B b broth

and incubated ovemight at 3TC and 225 rpm. A large plasmid prep of each of the

incubates (QIAGEN's Plasmid Mmci Kir) was then d e d out, followed by restriction

of 2 pg of each pEGFP-Nl/APP,,, construct with B a . 1. Reactioa mixture: 60

pl water, 8 pi bder , 2 pg DNA, 2 pl EcoR 1. The reactions were hcubated for 2 hr at

3TC, foliowing which, a 0.8% AGE was pedormed. To re-ver* the constructs, ten

more colonies were selected h m the pEGFP-N 1 /APP-T1m Ligation plate and added

to 5 ml L B h (50 pg/ml). These were incubated ovemight at 3TC md 250 rpm shake.

A plasmid miniprep (QIAGEN's Plamid Mini Kit) was performed on the incubates and

the DNA restricted sepanitely with EcoR 1 and BamH 1. Reaction mixture: 6 pl ddwater,

3 pl buffer, 1.6 pg (20 pl) DNA, 1 pl EfoR t or BamH 1. The d o n s were incubated

for 4 hr at 3TC. A 0.8% AGE showed similar overiapping supercoils. Thereforr, in order

to get a better separation of the DNA bands, the small AGE was p t e d with 1.6%

agarose.

3.1.6 pEGFP-N1/APP6W/N-TW comtruct

TO produce the shorter pEGFP-N 1 /APP695/N/NR08 ÇOnStnict (AFP,,, 1 -Zog),

the pEGFP-N l/APP695/N/NT,m COnstnict w ~ S d c t e d With Pst 1. This p d u c e d Linearized

pEGFP-Nl/APP,,n,,8, which was then d e d by Ligation. Reaction -: Four tubes

of 150 pl ddwater, 20 pl buffer, 50 pg pEGFP-NI/APP,,,,, 10 pl Pst 1. The

reactions were incubated ovemight at 3TC, following which, a 0.8% AGE was

performed (data not shown). The 4.9 Kb pEGFP-Nl/APP6951N-T#U) band was cut h m the

gel, and the DNA extracted by membrane electrophoresis. The DNA was precipitated and

purined via PCIAA and ethanol, as per Romega, and 1.5 pg of the pEGFP-NI/APP,,

T108~nstruct was Ligated using Boehringer-Mannheim's Rapid DNA Ligution Kif. The

protocol followed was as per kit, except that the ligation mixture was incubated for 1 hr

at room temperature-

To verify the constructs, pEGFP-Nl/APP695m5MT20d and pEGFP-NI weie restricted

with EcoR 1. Reaction mixture: 25 pl ddwater, 3 pl bu&, 1.7 pg DNA, 1 fl EcoR 1.

The reactions were incubated overnight at 3TC, foiiowed by 0.80/0 AGES.

3.1.7 pCMV/myc/mitoGFP p l u i i d

To ampli@ the pCMV/myclmitolGFP plasmid, 100 pl E coli DHSa cells were

tramformeci with 1 pg pCW/myc/mito/GFP plasmid, pmtocol as per Gibco BRL. AAer

the transformation procedure, the d s w e ~ re-suspended in 100 pl LB broth, and 50 pl

of this spread on an LBIamp plate and incubated overnight at 3TC. T h e colonies of

the E coli/pCMVlrnyc/mitolGFP were selected and added to separate vials of 7 ml

LB/amp broth. These were incubated for 5 hr at 3TC and 225 Tm. The contents of one

via1 were added to 500 ml LB/amp broth and incubated ovemight at 37% and 225 rpm.

A plasmid maxiprep ( QIAGEN's Plmmid M a i Kit) was perfonned on the incubate, and

the DNA wncentration determined photomeûically.

To verify the plasmid, the preparatïon was resîricted with Pst 1. Reaction mixture:

24 pl water, 3 pl buffet, 1.2 pg pCMVlmyc/mitdGFP, 1 pl Pst 1. The reaction was

incubated for 1 hr at 3TC, followed by a 0.8% AGE.

3.1 Libmry-de y#st twolhybrid msay

Am~lXcation of the vACTuh- brain çDNA ~lasmid ex~mssion librarv

The pACT2 Library contained 5 X 1 O' independent clones (or 1 O' colony forming

uni&) per d library. It consisteci of normal human brain cDNAs iaserted into the GAL-4

activation domain plasmid, pACT2 (Clontech). ùiitially, 4 pl (40,000 c h ) of the pACT2

library were spread onto each of two hundred 150 mm LBlamp plates, aod the plates

incubated at 30°C for 48 hr. On to each plate were s p d 5 ml LB/glycerol(25% v/v

glycerol), and the cells scraped into the liquid. AU the liquid was pooled and plasmid

maxipreps (QIAGEN's Plasmid Mmi Kir) performed to isolate the libraypACT2lbrain

cDNA.

To prepare sufficient yeast cells for the experhent, 2 0 pl of yeast strain Y 190

was spread on to a YPD complete yeast medium plate and incubated for 4 days at 30%.

YPD medium is composed of 20 g/L peptone, 10 g/L yeast extract, 20 g L agar and 2%

dextrose, pH 5.8, following Clontech's Matchmcïker Ga14 Two-Hybrid User Mmd

S e v d of the yeast colonies were scraped into 1 ml YPD medium. AAei vortexing, ceUs

were t r a n s f d to 150 ml YPD Liquid medium (same composition as YPD agay except

no agar added) and incubiited overnight at 30% and 250 rpm. 'Ibe incubate was

transferred to 1 L YPD liquid medium and incubated for 3 hr at 30'C and 250 rpm. The

celis were divided bto 50 ml tubes and centrifbged at 1,000 x g for 5 min. 'Ibe super-

natant was di&ed and the ceik re-suspended in 500 ml ddwater- The ceh werp

pelletecl (1,000 x g for 5 min), the supernatrmt discarâed, and cells re-suspended in 8 ml

1 X TE/ühium acetate (TE/LiAc). The TE/LiAc was made h m stock solutions 1 OX TE

b s e r (0.1 M Tris-HCI, 10 mM EDTA, p H 7.5) and 10X LiAc (1 M lithium acetate,

adjusteci to pH 7.5 with =tic acid [Sigma]).

Transformation of veast cells

To a tube were added and mixed 1 mg pAS2-l/APPTllY251U, 0.5 mg pACIZ/brain

cDNA, and 1 mg herring testes carrier DNA (Clontech). The herring testis DNA was first

denatured by placing it in a boiling water bath for 20 min and then immediately coolhg

on ice. To the DNA mi.mue was added the 8 ml of yeast Y 190 competent cells, and the

contents mixed by vortexing. Next, 60 ml of polyethylene giycoVLiAc (PEGLiAc)

solution was added and mixed by vortexing. Each 10 ml PEGKiAc solution was

wmposed of 8 ml of 50% polyethylene glycol (Sigma), 1 ml of 10X TE buffer, and 1 ml

10X LiAc. The transformation solution was incubated for 30 min at 30'C and 200 rpm,

fouowing which 7 ml dimethyl sulfoxide (DMSO) (Sigma) was added and gently mixed.

The solution was heat-shocked for 15 min at 42%. foliowed by chilling on ice for 2 min.

Next, the ceiIs were centrifbged for 5 min at 1,000 x g, and re-suspendeci in 10 ml 1X TE

buffet. The hansformed celis (200 pl) were spread on to each of fifty 150 mm SD/-Hid-

Leu/-TV/+ 45 mM 3-AT plates (i.e., a selection and phenotype-testing dropu t minimar

medium lacking ahno acids histidine [expressed by Y 1901, leucine [expresseci by pAS2-

11 and tqptophan [expressesi by pACT21, and with added 3-amhotriazole 13-AT] to

suppress a leaky His promoter), plus essentid nutrients such as a yeast nitrogen base,

dextrose, and a specific mixture of amino acids d nucleotides. The plates were

incubated at 30'C for seven days to allow good-sized colonies to develop. Forty colonies

(His') grew, which were candidates to contain both pAS2-1 and pACT2 placnrids.

@-naiactosidase Assay

To eliminate false positives, the coloaies that grew (His positive, His*) were

subjected to P-galactosidase colony-lift fllter assay, as per Clontech's M A T C W R

Yeasî Protocols Handbouk. For each plate of eaosformants to be assayed, a Whamian

#5 sterîle filter was pre-soaked in 5 ml of Z buffer/X-gid solution Z b u f k consisteci of

16.1 g/L N%mo4, 5.5 g/L NaKPO,, 0.75 g/L KCI, 0.246 g/L MgSO,, pH adjusted to

7.0. X-gal stock solution contained S-brom~hloro-3-indolyl-~-D-@actopyranoside

dissolved in NB-cisrnethyIformamide at a concentration of 20 mghi. Z buffer/X-gal

solution comprised 100 ml Z buffer, 0.27 ml p-mercaptoethanol, 1.67 ml X-gai stock

solution.

A clean dry filter was placed over the surf- of each plaie, and gently mbbed to

help wlonies cling to the filter. The flter was CU. to aUow for correct orientation. Afkr

the filter was evenly wetted, it was transfeRBd (colonies facing up) to a pool of liquid

niîrogen and completely submerged for 10 sec, afkr which it was removed and allowed

to thaw at m m temperature. The dried filter was placed, colony side up, on the pre-

soaked nIter, where it was incubated at 30'C untiI blue colonies developed in less than

8 hr. Eleven colonies (His' and LacZ) turned blue within 3 hr. These should contain

both the BD and ActD p l d d s . The correspondiag positive colonies were removed

h m the original plates and spread grid-iïke fashion on nesh SDl-Hid-Leu/-T@+ 45

mM 3-AT plates.

To put selection presswe on growth of colonies containhg AD/brain cDNA

constnicts only (i.e. select for colonies that have spontaneously lost BD/APP-,

constnrts), some ofthe His+RacZ+ colonies were re-pl& on SD/-Leu/+cycloheximide

(cyh) medium. Y 190 strain contains a gewmic mutant recessive cyh mistarit gene. The

BD vector expresses the wild-type dominant cyh sensitive gene, &le the ActD vector

contains neither gene. Therefore, only pACT-2/brain cDNA-contahhg colonies *ch

have no BD/APPnV3, constrwts will grow on the cyh+ medium. The pACT-Ubrain

cDNA clones were extcacted h m the yeast celis (by F. Amara) & stored at -20C for

futiae sequencing & protein analysis.

32.2 Small-scale yerst tnolhybrid amay

Vefication of the APP-,,-COX 1 interaction

Following Clontech's protocol, to make competent yeast ceiis, 1 ml ofYPD broth

was inoculated with several colonies of previously prepiued and h z e n (-80%) yeast

stmin Y190. M e r vortexing, it was transferred to 50 ml YPD liquid medium and

incubated overnight at 30'C and 250 rpm. The overnight culture was transferred to 300

ml YPD and incubatecl for 3 hr at 3WC a d 250 rpm. Fitty ml of the above culture were

added to a centrifuge tube and the ceiis pelieted (centrifiiged at 2500 rpm for 5 min). The

supernatant was discarded and the ceus R-suspended by voriexing in 25 ml watet. The

cells were pelleted again as before. The supema~aat was decanted and the now competent

ceils re-suspended in 1.5 ml 1X TE/LiAc-

To separate tubes were added 5 pg each of (i) pAS2-llAPPt7(VZSm, (ii)

pACm/COX I, and (iii) pAS2-11APP-, plus pACTZCOX 1. To each tube were

added 100 pl (1 mg) Herring testis caurier DNA and 300 pl wmpetent yeast cells. The

contents were mixed by vortexing- 600 pl PEG/LiAc were next added , the contents

vortexed and then incubated for 30 min at 30'C and 250 rpm- Next, 175 pl DMSO were

added and gently mixeci in. The tubes were then heat shocked for IS min in a 4 X water

bath followed by cold shock on ice for 2 min. The cells were peileted (cencnfiiged at

14,000 rpm for 5 sec), the supematant discardeâ, and ceils re-suspendeci in 200 pl TE

buffer. The DNA-transformed Y 190 suspension (200 pl) was spread onto SDl-Trp plates

for pAS2-lIAPP,-transformed ceiis, SDI-Leu for pACT2/COXl bansfomied cells, and

SD/-Leu/-Trpl-His/+45 mmo t 3-AT plates for pAS2- 1 IAPP, and pAC'T2-tnuisfonned

celis. The plates were incubated at 30% for 4 days to allow colonies to fully develop,

after which, a P-galactosidase colony-üft fiiter assay was perfomied on the HIS' colonies.

Small sale veast two-hvbrid assav with the APP deletion constnicts

The protocol was the same as that described above except that the tramdiormation

section involved adding to separate tubes 5 pg DNA coastructs ~AS2-llAPP-nom,

pAS2- I /APpmTl5,, pAS2-1 /APP--, P A S ~ - I I A P P ~ ~ ~ pAS2-1 l~PcT,-- . The

latter constnict consisteci of the APPfNNSî construct with the NHZ-terminal 2020 bps

removeci, and was prepared by B. Liang. To each tube were also added 5 pg

pACT2KOX 1. A P-galactosidase colony-lift fdter assay was perfonned on the above

colonies that grew.

3.3.0 Assays of APP SîabRy-hrnsfccted Ne~roblutoma CcM Linc

33.1 Neurobiastoma ceU cpltiiiu

The neuroblastoma SK-N-SH celis (ATCC) were gmwn in Neurobasal A medium

(Gibco BRL), supp1emented with 100/. fetai bovine senrm (Gl'bco BRL), 1 % L-glutamine

(Sigma), 1% gentamycin (Gi'bco BRL). Each I ml cnovial of quickiy thawed

neuroblastoma SK-N-SH ceUs was addeû to a sterile 50 ml centrifige tube, and to each

tube was slowly mixed 47 ml of wmplete Newbasal A medium. Next, 12 ml of this

neuroblastoma/culture medium mixture was added to separate culture flasks, which were

then Uicubated at 37% in humidified atmosphere ( 5% COJ95% air). The plasmids to be

used express4 the geneticin-resistant gene G418, which was used as a selection agent

to produce the stably transfected cell line. Cells were routinely passaged (trypsinlEDTA)

at 70-8096 confluence, and the medium changed as required.

33.2 Transfhon of neuroblrstoma ceU line

The transfection reagent used to transfect the neuroblastoma cells was

Boehringer-Mannheim's FuGENE 6 Trmfection Reagent, a blend of Lipids (non-

liposomal fornidation) and other components in 800? ethanol. The protoc01 foliowed

was basically that recomrnended by the manufacturer, as follows: The neuroblastoma

cells were subcuitured 24 hr before transféction, which e d they were 50-80%

coduent in the culture flasks. To 970 pi of medium was added 30 pl FuGENE 6

Transfecton reagent, and the mixture incubated at room tempetanue for 15 min. The

DNA (12 pg) was added to a second centrihge tube. Drop by drop, the diluteci FuGENE

6 reagent was added to the DNA. The tube was gedy tapped to mix the contents and

incubated at room temperature for 30 min. This uüxture was then added drop by drop to

the nemblastoma cells in 12 ml of medium in culture flasks, gently swirling the medium

to mix. The cells were then returned to the iacubator-

3 3 3 Isolation of mitocbondria from the neoroblastoma ctU line

The pmtocol was based on Vander Heiden et al. (1997). The medium was

vacuum aspirateci fiom the culture flasks growing the neuroblastorna celis, and then 5 mi

of 1 X ûypsin/EDTA added for 2-3 min. Next, 10 ml of wmplete medium was addeci, and

the cefis centrifuged at 2500 rpm for 5 min to pellet the cells. The nemblastoma cells

were re-suspendeci in 0.8 mi ice-cold Buffet A (250 m . sucrose, 20 mM KEPES, 10

mM KCl, 1.5 mM MgCl2, 1 m M EDTA, 1 mM EGTA, I mM Dm, 17 pg/d PMSF, 8

pg/rnl aprotinin, 2 pghl leupepth, pH 7.4), foiiowed by ultrasonic homogenization on

ice. The homogenate was cenîrifbged at 750 x g for 10 min to pellet uniysed ceils and

nuclei. The supernatant was centrifùged at 10,000 x g for 25 min. The resultant

mitochondriai peilet was ce-suspended in buffer A. The supernatant was centrifuged at

lûû,ûûû x g for 1 hr, the resulting supernatant behg the cytosolic f'ractioa-

3.3.4 cox activity assay in APP-tirnsfCCttd n t ~ r ~ b b t o a ~ ceU iiac

A reduced, approximateIy 5 mM cytochmme c solution was ppepared, exces

solid ascorbic acid was added and the mixtute allowed to stand for 10 min. A Sephamse

G50 column was prepared and equilibrated with 500 ml assay b@er (20 mM K,PO,, pH

7.0), any excess k i n g ailowed to drain. The reduced cytochmme c was pipenad into the

column, which was allowed to drain. More assay b a e r was added to the wlumn, and

the elution of cytochrome c foilowed visually, and collected in about 250 pi aliquots.

Prior to the COX assay the absorbante reading was checked at 550 nm, and

rechecked after the addition of sodium dithionate, to ensure cytochrome c was M y

reduced. For this assay, 990 pl of assay bu&x was added to a cuvette and the 4, set to

zero. To the b s e r 10 pl reduced cytochrome c was added and mixed by inversion. The

assay m . consisted of 50 pg mitochondrial protein, 0.2 mg dodecyl-maltoside (20

pl of a 10 mglm1 aqueous solution), 10 pl of reduced cytochrome c, and assay b a e r to

make 1 ml. The b d e r and dodecylmaltoside werr added to a cuvette, and the A,, set to

zero. Reduced cytochrome c was then added, and the cuvette equiiibrated at 30°C for 3

min. Next, the mitochondnal protein was added, and mixed twice by gentle inversion.

The absorbante was record& over 90 sec.

33.5 Comples 1 actmty u u y in APP-transf&ed neurobiastomi cell b e

The mitochondnal protein solution was diluted to a concentration of about 2

m g h l in sucrose-TRIS-ATP bufEer, and 200 pl of this was added to an eppendorf tube

and ultnwnicaily homogenized in ice. The assay was performed on 50 pg mitochondrai

protein. The assay volume was 1 ml, which consisted of 97 )il assay buffer (25 mM

K2P04, pH 8.40.25 mM K'EDTA, 1 mM KCN, 100 pM NADH). The buftk-containing

cuvette was warmed to 30'C for 3 min., and the reaction initiated by adding 3 pl of 20

mM ubiquinone in ethanol. The NADH oxidation was foliowed for 3 min at 340 nm.

The 1 min readùig was taken as average.

33.6 Detedon of apoptorh in APP-hrasf&ed neurobiastoma ce0 line

The neuroblastoma cells wne subcuinned on coverslips in weUs containing 2 ml

Neuralbasal A medium, When the ceils were 5&800/o confiuent, the cdture medium was

removed and the slips immersed in cold (2-CC) 1X phosphate buffered caline (PBS)

(Sigma). The incubation reagent was prepared and was composed of 10 pl of 1OX

binding buffer, 10 pl of propidium iodide (Geoyme) to check for cell necrosis, 1 pl of

AnneXia V conjugaie (Genzyme), 79 pl ddwater, to make a total volume of 100 (il. The

1X PBS wash was removed h m the slips, and exces liquid blotted away. 100 pl of the

incubation reagent was added to each sample, and incubateci for 15 min at room

temperature in the da& The cells were then washed twice for 2 min each wash with 50

ml 1X binding bufEer, foilowed immediately by fluorescence microscopy.

33.7 Assay for ROS kv& in APP-trriasfecteà neuabimtoma ceU üne

Cultures were washed with Dulbecco's modi£ied eagle's medium (DMEM)

(Sigma) and d o w e d to take up 10 pg (dichlorofluomscein diacetate @CF-DA) (added

as 10 nM stock in DMSO) for 1 hr. The cultures were washed twice with Hank's

buffered d h e solution (HBSS) (Sigma) and 5 pghl propidiun iodide (Geoyme) was

added to exclude debris and dead ceiis before data collection. The cultures were then

washed again with HBSS, md cellular fluore~cetlœ was viewed in HBSS using a Leitz

microscope with fluorescence nIters. In some cases, to provide standardized quantitation

of cell numbers, the nucleus-specific f l u o r n t dye Hoechst 33342 (Molecular Probes)

was added at 1 &ml at the tùw of addition of DCF-DA. Cultures were compared, with

stable transfectants and control conditions blind to the investigators. Fluoresence was

determineci quaatitativeiy by couuting h m nrst bright field mictograpbs, and then d e r

fluorescence rnicroscopy. Results were expressed as percent fluorescent cells.

The reaction mixture consisteà of 0 2 5 m M tert-butyl hydroperoxide, 1 m . GSH,

1.4 units glutathione reductase, 1.43 mM NADPH, and 1.5 mM KCN in a 100 mM

potassium phosphate buffer (pH 7.0) (Sigma). Volume = 1 ml - mitochoMina1

supernatant volume. tert-butyl hydroperoxide was used as substrate in order to measure

selenium-dependent glutathione pemxidase, the only isoform found in brain. Blank

values, obtained without addition of samples, were subtracted h m assay values.

33.9 Assay of superorkde d é m u f w acüvity in APP-transftcd neumbl.rtoma ceU line

Assay consisted of adding 25 jd of 24 m M pyrogallol in 10 mM HCI, 10 pl of 30

FM catalase (25,000 Ulmg), 50 pg mitochondrial protein, and assay b a e r (50 mM

TRIS-HCl plus 1 mM diethylenetriamine pentaacetic acid @H 8.2)) to make 3 ml

(Sigma), Absorbante was read at 420 nm. The blank containeci 25 pl pyrogallol, 10 pl

catalase, and 2.%5 ml buffkr-

3.4.1 TUT in vüro transcription-tmmhtion system

As supplied by Pmmega, the reaction mixture consisted of 25 pl rabbit

reticuiocyte lysate, 2 pl TnT d o n bunet, 1 pl TnT SP6 RNA polymerase, 1 pl of 1

mM amino acid mixture minus methionine, 40 microcurie @Ci) 3S~-methionine (1000

Ci/mmol at 10 mCi/rnl), 1 pl RNasin ribonuclease inhibitor (at 40 uCVml), 1 pg

pGEMS~APPm,,, IS pl RNA-fke ddwater. The ingmdients were mixed on ice and

the reaction mixture incubateci for 2 hr at 30'C. At the end of the incubation, 10 pl of

TnT reaction was then added to IO p1 of gel loading sample b a e r (50 rnM Tris-Cl (pH

6.8), 1 OYo glycerol, 2% sodium dodecyl d a t e (SDS), 0.1 % bromophenol blue, 100 mM

DIT) (Sigma), and the remainder stored at -20'C. The sample was heated at IWC for

2 min to denature the protein, and was then l d e d on to a 1W SDS-polyacxylamide gel

and subjected to electrophoresis (IV! SDS-PAGE), protocol and material as per

Sambrook et al., (1989), with Gibco-BRL's BenchMmk Protein Lodder as a m d e r (5

pl in 15 pl of sample bufEer). AAer the samples had nui the length of the gel, the gel

removed h m the apparatus. The portion containùig the protein ladder segment was

exciseà and fixeci and stained with Coumassie blue, as per Sambrook et al., (1989). The

gel was then dried and photographed.

The rest of the gel was p l a d on dry Whatmm 3MM paper, and then wrapped

in plastic wrap, and dned in a heated, vacuum drier. The gel was subjected to

autoradiography for 4 hr. A dominant main band ofjust under 100 kDa was seen, which

approxunates the expected size of APP. For the experiment, six TnT mictions were

performed and pooled at the end.

3-42 isoiation of rat mitochaadria

Mitochondna wete isolated h m rat bers based on Giuïivi et al., (1998). Two

rat livers (about 30 g total) were excised h m anesthetized rats and washed in 0.25 M

sucrose. Each iiver was homogeaized in about 50 ml MSHE buffer (0.25 M mannitol,

0.07 M sucrose, 0.5 mM EGTA, O. 1% fatty acid-f3ee bovine senun albu- (BSA), 2

mM K+-HEPES, pH7.4) (Sigma) at 4%. The homogenaîe was centnfiiged for 10 min at

6000 x g, and the resuiting supernatant centrifûged at 10,000 x g for 15 min to isolate the

mitochondrial pellet. To M e r purify and isolate the mitochondria, the pellet was re-

suspended in 5 ml MSHE plus 20 mi of 30% Pemll in 225 m M mannitol, 1 m M EGTA,

25 mM HEPES, 0.1% bovine serum albumin, and centrifiiged for 30 min at 95,000 x g.

The mitochonâria were collected h m the Iow dense yeliow-brown band. The

mitochondria were washed twice in MSHE at 6,300 x g for 10 min to remove the percoii.

The purified mitochondria were then washed twice in 150 mM KCI at 12,000 x g for 5

min, followed by two washes in MSHE at 12.000 x g for 5 min. The mitochondria were

fhaUy re-suspended in 1 ml MSHE, and the concentration of mitochondrial protein

detennined by the Bradford assay.

3.43 In vibo nit0ehondri.l import -y

The protocol for the classic mitochondrial import assay was based on Glick

(1992), Glick et 02- (1992), and Casari et al. (1998). To remove ribosomes, the TnT

reaction was centrifbged at 100,000 x g for 15 min. The import reaction was divided into

three different conditions: (i) 3sS-APPm plus mitochondria, (ii) 3 s ~ - A P ~ m plus

mitochondria plus trypsin, (fi) 3 S ~ - ~ P P m plus mitochondria plus min plus

valinomycin.

Reaction 1. 50 pl TnT reaction were added with 51.3 pg mitochondrial protein to 200

pl import buffer (0.6 M sorbitol, M n M K+-HEPES, pH7.0,50 rnM KCl, 10 mM MgCl,,

2.5 mM EDTA, 2 mM KH,PO,, 2 mM ATP, 1 m g / d BSA, fatty acid fke) (Sigma), and

incubated for 1 hr at 12"C, after which the reaction was terminated on ice.

Reaction 2. Two import reactions were run. Aîter the import reaction 0.25 m g / d and

0.5 m g / d trypsin (Sigrna) were added to the two separate reactions and incubated on ice

for 20 min. 1 mg/ml of trypsin inhibitor (Sigma) was then added and incubated on ice for

10 min.

Reaction 3. 1 pg/ml valinomycin (dissolved in ethanol) plus 50 mM KCl (Sigma) was

added to 5 1.3 pg mitochondrial protein in 200 pl import buffer and put on ice for 10 min.

20 pl TnT reaction was then added, and the import reaction allowed to occur as above.

This was foliowed by the trypsin and trypsin inhibitor treatments as above.

Samples nom ail four reactions were then centrifuged for 10 min at 12,000 x g

at CC, foliowed by resuspension in 100 pl import buffer and 1 mglml ûypsin inhibitor.

AU reactions were then cenirifüged at 12,000 x g for 10 min and re-suspended in 180 pl

import buffer. To this, 20 pl of Sû% trichlomacetic acid (TCA) (Sigma) was addeâ d

the mixtures heated at W C for 5 min. The samples were then put on ice for 5 min,

foiiowed by centrifugation at 12,000 x g for 5 min. The supeniataat was removed aad the

pellets re-suspended in 40 pl SDS-sample buffer. The samples, dong with the TnT

reaction as a standard and a Rainbow protein matker, were then subjected to 12% SDS-

PAGE. To deaease background radioactivity, after eiectrophoresis, the gel was boiled

for 5 min in 5% TCA, foilowed by a 5 min boil in water and then a 5 min soak in 1 M

Tris base. The gel was then vacuum-dried and subjected to autoradiography.

3.5 Tmtment of Nearoblrwtoma Ceb with MitoTncker Probu

The same protocol a p p k to MitoTracker Green FM and MitoTracker Red

CMXRos (Molecular Probes). The powdered dyes were first dissolveci in DMSO (Sigma)

to make a final concentration of 1 mM. Several concentrations were tested for best

resolution (20-500 mM) and it was found 20 mM or l e s to be most efficient.

The neuroblastoma cells were grown on coverslips in supplemented Neurobasal

A medium (as described previously) until50-80./0 confluent At this stage, the medium

was removed and replaced by pre-warmed (3PC) probecontaining medium, and

incubated for 45 minutes in the cell culture incubator. During the testing experiments to

detemine the optimum probe concentration, the cells were now examllied by

fluorescence microscopy. For the experiment proper, the cells were next nxed. Firsà, the

ceils were washed at 3TC with PBS. They were then fixed for 15 min at 37% using

fteshly prepared 3.7% para50rmadehyde in HBSS, following which, they were rinsed

several times with PBS. The ceh were then examined by fluorescence rnicroscopy, using

the appropriate filter sets for each probe.

3.6.1 Immunuelectron miemmpy to detect APP in mammrliin tissue

Uitrathin sections h m human and rat brain cortex and human kidneys were cut

h m tissue embedded in epoxy resin that had been fixed in KarnovsiqPs fixative

followed by pst-fixation in 1 % osmium tetroxide. Gold grid mounted sections were pre-

treated with 11% sodium metaperiodate for 1 hr. Mer several washes with dideci

water, the sections were blocked with a universai blocking buffet @AKO) for 5 min.

The sections were then incubatecl with mouse anti-human APP (Nb terminuus) antibody

(Zymed) (dilutexi 1 :5 in antibody dilution buffer PAICû]) for 90 mins. Control sections

were uicubaîed with anti-mouse immunoglobulin G (IgG) antibody, diluted 1 : 10. AAer

several washes in PBS, sectioas were incubated with goat anti-mouse gold-conjugated

IgG (diluted 1 5 ) for 5 hrs. Gold particle size was 10 nm (Cedar Lane Labs). AU

incubations and washes were conducted at room temperature. Foilowing several washes,

the sections were fixed in glutaraldehyde and treated with 3% uranyl acetate for 5 min.

prior to visuaikation- Labeiied sections were viewed with a Phillips 201 transmission

electron microscope, ancl representative areas were photographeci.

3.62 I m r n p 1 1 0 c l ~ n mNr(wcoj~~ ta deteet APP and BA' h human b&

Ultrathin sections h m temporal cortex of AD (n = 4) and non-AD patients (n =

4) (National Nemlogical Research Specimen Bank, VA, Medical Centre, LA) were cut

h m tissue embedded in epoxy resin that bad been fhed in Karnovsky's kative

followed by post-fixation in 1% osmium tetroxide. Nickel grid mounted sections were

pie-treated with 1 1 % sodium metaperiodate for 1 hr. Mer several washes with distilleci

water, the sections were blocked with a universal blocking b&er (DAKO) for 5 min.

The sections were then incubated with mouse anti-human APP (Mi, terminus) anti'body

(Zymed) (1 :5 dilution in antibody dilution bufEer [DAKO]) for 90 mins. ContFol sections

were incubated with anti-mouse IgG antibody, dilided 1 5. Afkr several washes in PBS,

sections were incubated with goat anti-mouse IgG pld-conjugated antibody (diluted 1 5)

for 2 hrs. Gold particle size was 10 nm ( C e . Lane Labs). AU incubations and washes

were conducteci at room tempera-. Followiag several washes, the sections were fixed

in glutaradehyde and treated with 3% uranyl acetate for 5 min prior to visualhtion.

Labeled sections were viewed with a Phillips 20 1 transmission electron microscope, and

representative areas were photogaphed, Similarly, PAP was visualued using rabbit anti-

human PAP antibody (Zymed, dilution MO) , and gold wnjugated (5 nm), goat anti-

rabbit IgG (British BioceU, dilution 1 50).

The APP constructs that were used as bait proteins in the yeast two-hybnd assay

were prepared as describeci. First, the pAS2-1/APPmI# construct was prepared. To

ver@ the originating ~GEM.~zVAPP-~~ wnstnict, it was resfficted with Bgl ii.

A.PPm3* has one Bgl II site at bp 1994, wbiie the plasmid pGEM9zîhas no Bgl II sites.

The one DNA band of about 6.5 Kb was seen on the agarose gel (data not shown).

To isolate the APPmm fragment for insert into the pGEMSzf plasmid, the

pGEM9zDAPPnm, construct was restricted with Spe 1. APP-, has one Spe 1 site at

bp 2583, while pGEM9zfhas an Spe 1 site in the MCS NH2-terminai to the APP insert.

Thus, there were two bands of about 3 Kb (pGEM9zfplasmid) and 2.58 Kb (APP-,)

(data not shown)..

The pGEM5zfplasmid was then restricted with Spe 1 to prepare it to accept the

Spe 1 -restricted APP-, fhgment. The pGEMSzf plasmid has only one Spe 1 site in

the MCS, and therefore the= was only one band of 3 Kb seen on AGE (data not shown).

To verify the correct orientation of the N P m a insert Mer ligation into the pGEMSzf

plasmid, the pGEM5zUAPPma consûuct was restricted separately with Sac 1 and Pst

1. Sac 1 has one site in APPma at bp 1813, and one site in the MCS of pGEMSzf.

Therefore, for Sac 1 -resbricted pGEMSz£(APPma, correct orientation produced two

bands of 0.8 Kb and 4.9 Kb, and the incorrect orientation produced two bands of 1.85 Kb

and 3.86 Kb. Pst 1 has four sites in APP-,, at bps 208,238, 1438, and 1655. It bas

one site in the MCS of pGEM5zf. Therefore, for Pst 1-riestncted pGEMS~APPmm,

the correct orientation produced four bands visible of 0.22 Kb, 0.95 Kb, 1.2 Kb and 3 -32

Kb., while the incorrect orientation produced four bands visible of 0.22 Kb, 0.23 Kb, 1 2

Kb and 4.04 Kb (data not shown).

To pnqme the APP,, insert and the GAL4 binding domain (BD) pliasrnid,

pAS2-1, to receive the iasert, both the pGEMSzVAPP-, wnstruct and pAS2-1, were

simuitaneously restricted with Nco 1 and Sa1 1. There is one Nco 1 and one Sa1 1 site in

the MCSs of each plasmi6 In the pGEMSzfplasmid, the Neo 1 site is 5' to the Spe 1 site

(and therefore the APP insert), while the Sa1 1 site is 3' to the Spe 1 (and therefore APP

insert) site. Therefore, for Nco l/Sal l-restricted pGEMSzf7APPm, there were two bands

of 2.66 Kb (APPnmU) and 3 Kb (pGEMSdplasmid). For the Nco 1/Sal 1-restricted

pAS2-1, there was one band of 8.4 Kb (iinearized plasmid) (data wt shown).

Afiet Ligation of APP-, into pAS2-1, the constnicts were verified by

restrïcting pAS2-l/APP-, in separate tubes with Pst 1, Bgi Ii or Hiod m. Pst 1 - restricted pAS2- l/APPmm showed six bands of 0.003 Kb, 0.0 18 Kb, 0.03 Kb, 0.22 Kb,

0.93 Kb and 1.2 Kb. Bgl II bas one site in pAS2-1 at bp 3969. and one site in APP-,

at bp 1994. Therefore, Bgl II-restricted pAS2-l/APPmM showed two bands of 4.56 Kb

and 6.43 Kb. Hiid UI bas four sites in pAS2-1 at bps 2397,3284,5477,6224, and no

sites in APP-,. Therefore, h d III-restricted pAs2-l/APPTIOlU83 demoostrated four

bands of 0.89 Kb, 2.1 9 Kb, 3.36 Kb and 4.57 Kb (data not shown).

Eukaryotic transcription factors (TF) contain two physicaily and h c t i o d y

seprable domauis - an NH,-terminal DNA-binding domain and a COOH-terminal,

acidic activation domain. The BD specifically binds to a promoter or other cis-regdatory

element. The ActD directs RNA Polymerase il to traascribe the gene do- of the

DNA binding site. The physically divided TF can hinction wrmaUy, as long as a bridge

is provided that localizes the ActD to the BD bound to the promoter. The bridge is

formed by the interaction of one protein expressed as protein X (e.g APP) füsed to a BD

with another protein expressed as protein Y (e-g brain cDNA) fused to an AD, where

proteins X and Y norrnally interact in cells. (Figure 6). In the yeast assay, an interaction

between two human proteins brings together the modular BD and ActD of the yeast

GALA TF, which causes the activation of transcription of a reporter gene in the yeast

host.

The yeast seain used in the assay, Y 190, has two reporter genes, LacZand HIS3,

as separate constructs integrated into its genome. The Lad gene encodes the bacterial

enzyme beta-galactosidase, which causes colonies to tum blue when exposed to a

specific substrate. The HIS3 gene encodes an enyme in the histidine biosynthetic

pathway, and acts as a nutritional marker for yeast cells grown on histidinedeficient

medium. As weil, Y190 is auxotrophic for tryptophan and leucine, two amino acids

encoded by the BD and ActD plasmids, respectively. This ailows selective growth by the

yeast C ~ U S on histidine-, tryptophan- and leucinedeficient medium by only those yeast

Figure 6 Schematic diagram of the MATCHMAKER GALA-based two-hybrid systems. (Adapted h m Clonteîh catalog).

ceils transfected with both plasmids (Figure 7).

Figure 8 summarizes the f'indings of the sc&g of the human brain cDNA

expression library. Screening of ld double traasformants resulted in 40 HIS3 positive

colonies, of which 1 1 were subsequently shown to be both HE3 and L a 2 positive.

4.2 Analysis of Positive Clona

Two clones (A-2 and A-14) that showed the strongest protein-protein

interactions, ôased on the speed and intensity of the ~galactosidase blue wlor

development, were selected for fkher analysis. They were sequenced at the fûsion site

and shown to be in-frame with the GALA-pACT2 ActD. A-14 cornpriseci 680 bases with

an open resding frame endhg at 606, encoding a protein of 202 amino acids, A-2 was

of the same gene but longer by 938 bases.

To identify homologous proteins, the BLAST data base of the National Centre

for Biotechnology Information was searched. Both cDNA clones shatod over 99%

identity with the f d y of cytochiome c oxidase enzymes of the mitochondrial ETC.

Sequence d y s i s (Devereux et al., 1984) of Match clones identifsed them as complete

and partial COX subunit 1 gene sequences coupled to ~RNA* (Anderson et al., 198 1).

A-14 was found to be identical to the 1st 606 bps of COX 1 plus the adjacent (in the

rnitochondrial gemme) ~RNA*, while A-2 was composed of the whole COX 1 sequence

attached to tRNAScr. The A-2 COX 1 gew sequence comprised bps 5899-7444 of the

mitochondrial genome, while A 4 4 consisted of bps 6837-7444 (see Figure 9).

Figure 7 Screening an AD fusion library for proteins that interact with a bait protein. (Adapted h m Clontech cetalog).

106 double transformants screened

40 colonies, His+

1 1 colonies, His+ and L a d +

Two clones, A-2 and A-14 were independently isolated. These clones encode wmplete and p h a l cDNA sequences of human brain

mitochondnal cytochrome c oxidase subunit 1 (COX 1) wupled with the wmplete cDNA sequence of ~ R N A ~

Figure 8 Matchmaker two-hybnd screen. pAS2-IIAPP was used to screen a whole human brain cDNA library. Among the interacting clones, A-2 and A44 encode sequences of COX 1 and WUA* (Anderson et al., 198 1).

To establish that the interaction of APPmm and COX 1 was specific, yeast Y 190

ceus were transformed in separate experiments with pAS2-I/APP-, alme,

pACTUCOX I alone, and the two constructs together, using Clontech's Mutchmder

Ga14 Two-Hybn'd User Mamai for SmaU-de transformation, followed by Clontech's

p-gaiactosidase coiony-iift filter assay. Neither pAS2-11- nor pACT2/COX 1 by

themselves activated P-galactosidase activity, indicating that wither APP, nor COX I

contained a latent transcriptionai activaîor- Only when the GAL4-BD/APPma3 gene

was CO-transformeci withthe GALA-ActD/COX I did activation ofthe LacZreporter gene

occur. Thus, it appears APPma3 and COX 1 do not interact non-specindy with other

proteins using the yeast MTCMCLQKER system. The specificity o f the APP-COX 1

interaction was also confirmed by Amara and Junaid (manuscript in revision) by the lack

of P-galactosidase activity when the APP-related constructs or COX 1 were expressed

alone, or when APP,,, was co-expressed with COX ï I and COX III, d e the three major

isoforms of APP interacte- with COX 1 (Table 1).

4.4.0 Preparation of APP Dektion Cowtructs

To determine wbat general region of APP, is involved in the interaction with

COX 1, REs were used to delete portions of the APP, cDNA, as described. The

resulting constnicts were tested for interaction with COX 1 usiag the MATCHUlKER

Table 1 Interaction of APP and COX 1 in yeast. Amyloid precursor protein plasmids (pAS2- I/APPs) cloaed into a GAIA DNA binding domain fusion vector were tested for their ability to interact with COX 1-GAL4 activation domain fusions. Positive resuits were measured as the developxnent of blue color on X-gal filter iifts of colonies expressing both activator and DNA binding wnstructs relative to colonies expressing each consûuct alone. Filter resuits (column 4) were confinned by quantitative iiquid culhire assay (column 3). Negative resuits (white colonies) were obtained for the interaction between GALA-binding d~rnain/APP,~, and GAIA-activation domaidCOX II and COX III. (Courtesy Amara and Junaid [manuscript in revision]).

yeast two-hybrid assay and p-galactosidase colony-lift flter assay.

4.4.1 pAS2-1/APP2020 ~001~trPct

The pAS2-1/APP-ma constmct has EcoR 1 sites at bps -15 of the 5' UTR of

APP and at bp 2020 of APP. Therefore, EfoR 1 restriction removed a segment of APP

h m PAS~-I/APP, ,~-~, that containeci the first 2020 base pairs of APP plus 15 bps

of the 5' UTR. Two bands of 9 Kb @AS24 plus COOH-termiml APP) and 2.035 Kb

(the APPîMM,, band, cumprising NP,, bps 1-2020) were seen on the agarose gel

(Figure 10). This NH,-terminal APP segment was inserted into the pAS2-1 p l d d to

mate the construct, pAS2-l/APPmm.

To vem the correct ligation and orientation of the APP710M710MT2020 fhgment ht0

pAS2-1, the pAS2-I/APPl10/N-lZOM constructs were restricted with Sac 1. The pAS2-

llAPPnm-nmo constnict has two Sac 1 sites: one at bp 3298 of pAS2-1, and one at bp

1813 of APPnWnO2p Therefore, correctly oriented constructs produced two bands of

4.52 Kb and 5.92 Kb, and incorrectly oriented constructs showed two bands of 0.9 Kb

and 7.75 Kb (data not shown). To re-ve* the correct constnict, the pAS2-l/APPm

,, preparation after amplification was restricted with Sac 1. The 4.52 Kb and 5.2 Kb

bands were seen (Figure t 1).

pAS2-l/APPnarm- and pAS2-1/APPm-- eonstrucb

To create smaller APP deletion consttucts, pAS2-l/APP-m was restncted

separately with BamH 1 and Pst 1. The pAS2-1/APP7M/N-TZm construct has two BamH 1

Figure 10 Restriction enzyme digestion of pAS2- IIAPP ,,, with E w R 1. Laue 1. Unrestricted pAS2-l/APP ,,,,. Lane 2. 1 Kb DNA ladder (GibcoBRL). Lanes 3-7. EcoR 1-restricted pAS2-l/APP nwUa. DNA stained with ethidium bromide in 0.8% agarose gel.

Figure 1 1 Sac 1 restriction euyme digestion of pAS2- I/APP,N-E~2,, consüuct. Lane 1. 1 Kb DNA ladder (GibcoBRL). Lane 2. Unrehcted pAS2-l/APP,m--Em. Lanes 3 & 4. Sac 1 -restncted pAS2-l/APP,,wN-nao. Verifkation that conamct was in correct orientation. DNA stained with ethidiurn bromide in 0.8% agarose gel.

sites: one in the MCS of pAS2-1, COOH-terminal to the APP insert, and one at bp 1554

of the APP insert. Therefore, the BamH 1-restricted pAS2-l/APP-,, yielded two

bands of 0.47 Kb and 9.97 Kb (data not shown). The pAS2-1/APPTIOIN-'12020 coastnrct has

five Pst 1 sites: one in the MCS of pAS2-1 COOH-terminal to the APP insert, and four

sites in APPm-nmo at bps 208,238, 1438, and 1655. Therefore, the Pst I-restrïcted

pAS2-l/APPm-no, yielded five bands of 0.03 Kb, 0.2 Kb, 0.4 Kb, 1.2 Kb and 8.64 Kb

(datanot shown). BamH 1 produceà PAS~-I/APP-~,~ (APP, uisert containing bps

1 - 1 5 54), and Pst 1 produced pAS2- l/APP710/N-'IM(I (APP, insert containing bps 1 -208).

AAer iigation of the pAS2-I/APPn[)/N-R08 and pAs2°1/APP'TMIN-Tlm constructs,

they were v d e d using Hind III restriction. In both coflStr\lcts, Hind III has no sites in

the APP inserts, but has four sites in pAS2-I at bps 2397, 3284, 5477 and 6224.

Therefore, the Hind III-restricted pAS2-l/APP-'IZw wnstruct yielded four bands of

0.89 Kb, 0.94 Kb, 2- 19 Kb and 4.57 Kb. The &d m-restricted pAS2-1/APP710/N710/NT,,

construct p d u c e d four bands of 0.89 Kb, 2.19 Kb, 2.3 1 Kb and 4.57 Kb (Figure 12).

4.4 Yeast Two-hybrid Assay With APP Dektioa Coafitructs

To determine with which APP constmcts COX 1 interacts, and thus determine

what generai region of APPm is involveci in the interaction, a srnail scale yeast two-

hybrid assay was performed, followed by a f3-galactosidase wlony-lüt £üter assay. AU

coastnicts were positive except for pAS2-l/APPCTIIN-m. This suggests the APP-COX

1 interaction involves the nrSt 208 N H 2 - t e ~ nucleotides of APP,. These results

Figure 12 Huid III restriction o f constructs pAS2- I/APP,,,, & pM2- l/APPnas- ,,,,. Lane 1. 1 Kb DNA ladder (Biorad). Lane 2. Unrestricted pAS2- l/APPnaw-p,8. Lane 3. Hïnd III-restricted pAS2-1/APPnm--. Lane 6. Unrestncted pAS2- l/APPnO/s-T, ,,. Lane 7. Hind III-restricted PAS~-I/APP~~-~,~,. Lane 8. High molecuiar weight DNA marker (J3iorad). The two constnicts were verified. DNA stained with ethidium bromide in 0.8% agarose gel.

were supported by a repeat small scale yeast two-hybrid assay- Plasmids examined were

pAS2- 1 /APP77WN-77,020, pAS2-1 /APPma, and p ~ 2 - 1 / ~ P C C T I 1 2 0 2 0 , with pACT2

added, plus pAS2-1 fAPPmm alone. Only pAS2- l/APP-nao and pAS2- L/APP-,

showed signifiant values for P-gaiactosidase activity (data not shown). This confümed

that at Ieast the NH,-terminai 2020 amino acids are involved in APP's interaction with

cox 1.

43.0 APP and Mitochondrh

For APP to interact with a facultative mitochondnal protein, we postulate that the

interaction must take place in the mitochondria, and that therefore, APP must be targeted

to, and localized in, mitochondria.

4.5.1 Mitochondrial targcting signal

If APP is targeted to and localizes in mitochondria, it should contain a

mitochondrial targeting seqiience. Cornputer anaiysis ofthe APP leader peptide sequence

using the PSOR-T II program (see Iinpr//psorttnibb.acJp/c~-bidoAumwu), suggested the

fkst 1 7 amino acids are a possible mitochondrial targeting sequeme (MiTDISC), and that

the cleavage site for the mitochondrid pre-sequence occurs between residues 17 and 18

in the sequence ARALEV (ûavel). These results were supported by the Si# (see

hnprgenome. cbs-dru- dMtbidnph-webfuce) program whkh suggested APP has a signal

sequence wnsisting of the first 17 amino acids. These hdings are consistent with a high

probability of intra-rnitochondrial sorting of APP (Gave1 and von Heijne, 1990). Based

on this anaiysis, we tested our hypothesis tbaî APP resides in mitochondria and under

certain conditions, impairs the activity of COX, enhances the production of ROS and its

attendant lipid peroxidation, a d induces apoptosis in mammalian celis, specifically, a

neuroblastoma ceil line.

To examine the d e of APP overexpression in AD, SK-N-SH neuroblastoma

cells were stably transfected with various APP constructs, and the following parameters

were examined; (i) complex 1 and complex N enzyme activities of the ETC, (ii) levels

of ROS production and lipid peroxidation, (üi) mti-oxidant enzyme activities of

superoxide dismutase. and glritathione peroxidase, and (iv) the degree of apoptosis

induceci. The pShooter expression vector, pCMV/myc/mito, which expresses a

rnitochondrial targeting sequence, was used as the transfectiog vector to sufcessfblly

express APP in mitochondria h addition, the expression vector SI-ne0 (Romega),

which ailows generalized expression of APP, was aiso employed by Amara and Junaid.

The effects of targeting APP to mitochondria on the various parameters

mentioned were determineâ by inserthg APP-, cDNA into pCMV/myc/mito to

create the constnict pCMV/myc/mito/APPnanstU. To isolate the APPmm -en& the

pGEMSzVAPPnW, construct was restricted with Spe 1. The construct has two Spe 1

sites: one 39 bps 5' to the amino terminus of the APP insert, and one at bp 2583 of the

APP insen Therefore, the Spe I-resfricted pGEMZzyAPP-, producd two bands of

2.62 Kb (the APP-, inrat) and 3 Kb (pGEM5zfplasmid) on an agarose gel(data mt

shown).

The pBluescript II SK was next restricted with Spe 1 to d o w it to accept the Spe

1 -restcicted APP-, hgment There is o d y one Spe 1 site in p B l u e p t II SK in the

MCS, which was seen on AGE (data not shown). Mer ligation of the NP-,

m e n t hto the pBluescript II SK phagemid to create the pBluescript II SWAPP-,

constnict, the correct orientation of the insert was verified by RE analysis (data not

shown).

To prepare the pCMV/myc/mito plasmid to accept the APPmm insert, it was

simultanmusly restricted with Sa1 1 & Not 1 REs. There is one site each for Sa1 1 and

Not 1 in the MCS of pCMV/myc/mito. Therefore, two bands of 0.02 Kb (MCS segment)

and 4.94 Kb (Sal 1 Mot 1 -restricîed plasmid) were present on AGE (data not shown). The

pBluescript II SWAPPnOm, construct was also restricted with Sa1 1 and Not I to field

the NPnm, fragment, which was Ligated into the Sa1 INot 1-restricted pCMV/myc/mito

plasmid to create pCMV/myc/mito/APPm. This constnict was venned by RE

analysis (&ta not shown).

4.6.1 Assays of COX and Compkx 1 eaeynatic activitia

COX actlvitv assav

The assay involved foiiowing the oxidation of cytochmme c, and the protoc01 was

based on Parker et al. (1994 b). Activity was calculated as a fjrst order rate constant,

calculaiions king based on the equation:

Velocity = k[cyt cl phUsec,

where k = ntst order rate constant = 231og bW (at time O) /A,,

(at time O + time 1 min) min-'.

The cyîochrome c concentration was d e d d by the equation:

[cyt. c] = 4, / 0.029.

(Extinction or absorbame coefficient for cytochrome c is 29m.M-')-

Com~lex 1 rctivihr asmv

To compare the effets of APP on other enzymes of the ETC, the activity of

Complex 1 was also assayed by followiag the oxidation of NADH. Calculations were

based on the following:

(i) The molar extinction coefficient of NADH is 6.81 mM-'.

@) WHJ = A, / Ext. Coef.

(iii) Rate = initial NADH] added (1 00 pM) - [NADHI at tirne 1 min (pM/min).

The assay was then ~peated in the presence of 2.5 rotenone, which inhibits

Complex 1 acîivity- Final rate was calculated as that without rotenone - thaî with

rotenone.

. - 9

There was a markedly reduced level of total COX activity in the cells

overpxpressing mitochondria-targeted APP compaRd with other transfectants (Table 2).

Complex 1

moi min-'S.E.M.

Complex iV

Sec"/mg pmteinkS.E,M.

Table 2 Complex 1 and wmplex N enzyme activities in nemblastoma cells overexpressing APP. The specined values represent the meam of complex 1 and Complex N activities +/- SEM. of ten replicates. Activity was measured by spectrophotometry. The human neuroblastomaceIls were stably transfected with différent plasmid constructs in contrast to untransfected cells.

in con- no signifiant change was observeâ for cornplex 1 activity between the SK-N-

SH transfectants It is noteworthy that nuclei-targeted APP or mitochondriatargeted a-1

antichymotrypsin (a PAP plaque-associated protein) overiexpression did not signïfïcantly

aftèct COX activity in SK-N-SH stable traosfectants (datanot shown). This suggests that

the accumulation of APP in mitochoodna plays a crucial d e in specifidy mediating

decreased COX activity.

4.6.2 APP overexpression and apoptost in nerrobîastoma ceU l i ie

Using Genzyme's TACSAmain VAppiosis Detection Kit, there was found to

be a prominent rise in apoptosis in cells traasfected with pCMVlmyclmtolAPP

(Mitochondrial expression of NP), compared with either vector alone

(pCMVlmyclmio) or mtransfected ceiis (Figure 13). In contrast, there was no signifiant

difference in the extent of apoptosis in cells transfated with $1-neo/APP (generalized

cellular expression of NP), compared with vector alone @CI-na) or untransfected cells

(data not shown) (Amara and Junaïd [manuscript in revision]). These d t s suggest that

overexpression of APP on its own, may not be sufncient to induce apoptosis in neufons

without the accumulation of APP in mitochondria.

4.6.3 APP overerpnssion and ROS production h neuroblutoma di iine

TO determine the d e of ROS in APP-mediated neuronal apoptosis, it was

examined whether neiuoblastoma cells targeted with overexpmssion of APP wodd

generate increased Ieveis of ROS. To detect ROS, the non-fluorescent dye 2'7-

Figure 13 Targeting APP into mitochondna potentiates apop tosis. SK-N-SH ceUs were a. untransfkcted, b. stably expressing pCMV/myc/rnito, and c stably expressing pCMV/myc/mto/APP. Plasrnid consmicts were assayed for induction of percentage apo ptosis by Annexin conjugate accord'ig t O the manufacturer' s instructions (2 ymed) . In apoptotic assays the percentage of necrotic ceiis was negligible (-%). Late apoptotic cells were stained redgreen. Scale bar: 100 Pm. d. Percentage apoptosis index. The mean +/- S.E.M. of 10 replicates as calculateci for >200 cells is shown. Asterisks: P, 0.0 1. The images were enhanced in size 2.0-4.0 fold. Ceiis were transfected using FuGENE 6, according to the manufacturer's instructions, and stably selected in 600 G4 18 (Invitrogen) .

dichlorofluoroscein diacetate @CF-DA)was used, which interacts with intraceiluiar ROS

to generate the fluorescent 2,T-dichlorofluoroscin proâuct (Rozenkranz et al,, 1992).

Fluorescence mimscopy demonstrateci no significant diBTerence between levels of DCF

in contml cells tmnsfected with pCI-ne0 and pCI-nedAPP (Figure 14 a,c). In con-

there was markedly increased ROS generation in pCMV/mydmito/APP stably

tcmsfécted cells compared with pCMVlmydmit0, and with untransfected ceUs (Fi-

14 b,d).

4.6.4 APP overexprrsrion and the formation of ïipiü pe ro~ t ion

To confirm that i n d fkee radiai generaîion resulted in cellular damage in

pCMWmyc/mito/APP transfectants, lipid perorùdation based on loss of fluorescence

nom inçorporated cis-parimric acid (CPA) was detemined. Lipid peroxidation in pCI-

NeoIAPP cells was not sigdïcaatly different than that detected in normal wntrol or SK-

N-SH pCI-neo transfectants (Figure 14 e), but was signifïcantiy greater in

pCMV/myc/mito/APP SK-N-SH transfectants compared with pCMVlmyc/mito

transfectants and untransfected SK-N-SH cells (Figure 14 f). These results suggest that

a defect in metaboiism of ROS in mitochondria-targeted APP o v e r e x p d SK-N-SH

celis leads to apoptosis.

4.6.5 Anti-oMluit enzyme .ssriys

To assess the possibility that exthanced ROS production may be due to an APP-

mediaîed effect on ROS scavenger enzymes, the activation of k e radical scavenging

Figure 14 Induction of ROS and Lipid peroxidation in SK-N-SH ceils. a. SK-N-SH cells (I) untransfected, stably expressing (II) pCI-neo and (III) pCI-neo/APP were exposed to 1 0 pg 2', i',dichlorofluoroscein diacetate for 1 hr. The cells were washed with HBSS and viewed with a fluorescence microscope ushg fluoroscein optics. Arrow heads indicate some of the intraceiiularly appearing fluorescence. Scale bar: 100 Pm. b. (I) SK- N-SH cells untransfected, and stably expressing (II) pCMV/mydmito, and (IU) pCMV/mydmito/APP were treated with DCF-DA as describeci above. Scale bar: 1 00 Pm. Fields were chosen to iiiustrate represeatative fluorescence micrographs. c/d. Values are expressed as percent of the DCF level in normal cultures ( 10W) and represent the mean +/- S.E.M.. n > 400 cells. df. Lipid peroxidation is indicated by the percent decrease in cis-parinarie acid (CPA) fluorescence. Values are the mean +/- S.E.M.. n > 400 cells. Asterisks: P c 0.00 1. The images were enhaneed in size 1-5-2.0 fold.

enzymes, glutathione peroxidase and superoràde dismutase, were examined in the

neuroblastoma celis.

Glutathione ~roxidase w ~ v

Glutathione peroxidases catalyze the reduction of hydroperoxides (ROOH) by

glutathione (GSH):

ROOH + 2GSH - ROH + H,O + GSSG

GSSG is reduced by the reaction catalyzed by glutathione reductase:

GSSG + NADPH + W - 2GSH + NADP

Glutathione pemxidase activity was measured at 37% by a wupled assay system

in which the oxidation of reduced glutathione (GSH) was coupled to NADPH oxidation

catalysed by glutathione reductase, following a pmtocol based on Carrnagnol et al.

(1983). Specific activity was determined as the amount of enzyme that catalyzed the

transformation of 1 nmol of NADPWinin. Rate = m o l NADPH transfonned /min-

Su~eroxide dismutase amav

SOD activity is assayed by tracking the inhibition of pyrogallol autooxidation.

The protoc01 used was based on Mariclund (1985). Calculations were based on the

equation:

% inhibition = (A A sample - A A p p g d o l ) I A A pyrogallol

where pyrogallol gives a A A = O.OUmin,

Resdb

APP overexpression had no significant effect on the activity of glutathione

peroxidase (Cannagnol et d., 1983) in the stably îransfected (Figure 15 a) cells

compared to wntrol ceiis, but there was a signifiant increase in SOD actiMty (Nagï et

al., 1995) in the pCMV/myc/mito cells compsired to other transfectants and conttrol cells

(Figure 15 b). Thus, only mitochondna-targeted APP once again had an effect. This

action m a . be a sign of a compensatory response by the enzyme to increased ROS

production.

4.7.0 Evidence for the Physiologhl Locrition of APP in Mitochoadria

For APP to interact with a mitochondrial enzyme, APP m u t be targeted to and

locaiized in mitochondria. To examine this, three experiments were performed: (i) a

classic in vitro mitochondrial import assay, (ii) fluorescent microscopy of neuroblastoma

cells transiently transfected with chimeric APP-GFP constructs, and (iii) immunoelectron

microscopy of brain tissue h m AD aud control patients to visualize the location of

endogenous APP and PAP.

4.7.1 Classic in vrtro niit0choadri.l import aaaay

The aim was to show that 3sS-methionine-labelled APP is imported into

mitochondria (isdated h m rat livets) in an enegydependent manner. It is known thaî

proteins to be imported into mitochondnal are synthesized in the cytoplasm in a

Figure 15 Comprison of the fke radical scavenging enzyme activities. a. GluSethione pekxidase, and b. Superoxide dismutase (SOD) in SK-N-SH nemblastoma cells stably k f e c t e d with different p l d d constnicts and control celis (mtransfected).

precutsor form, whose mitochondrial targeting sequence is cleaved after import,

generatùig one or more shorter mature f o m . The experimentd conditions allow APP

to be importeci into mitochondria (if it bas a mitochondrial targeting sequence). The

mitochondrial preparations are then treated with trypsin to completely digest any

unimportecf APP. This will determine if any APP was imported into mitochondria, and

therefore qared nom typsin proteolysis. It wili also aüow d e t e o n of the

approximate size of the imported hgment (s) and therefore, show whether a portion

(e.g., targeting sequence) has been cleaved off within the mitochondna in a separate

experhent, the import reactions were dso pre-treated with vaihomycin, a potent

inhibitor of COX. Because valinomycin destroys the ability of mitochondria to produce

any energy? no APP should be imported into mitochonâria, ifsuch importation is energy-

dependent.

Therefore, fiom the experimentai setup, expected bands include: (i) TnT product

( 3 5 S - ~ ~ ) lane. One band representiag the precufsor APP,,,,. (fi) Mitochondrial import

reaction without typsin treatment (NP, plus rnitochondria lane). Two or more bands

representing preciusor APP, plus one or more mehire shorter forms of APP,, with the

mitochondnal targeting sequence cleaved off. (5 ) Mitochondrial import reaction

followed by trypsin treatment (APP, plus mitochondria plus trypsin lane). One or more

mature bands, but no precucsor APP, band. The APP within the mitochondria is

inaccessible to the trypsin, whereas the precuisor APP is accessible and Mly cleaved.

(iv) Pre-treatment of import reaction with vaünomycin (APP, plus mitochondria plus

trypsin plus valinomycin lane). No bandsy as there is no energy to import APP. This

shows that the import of APP into mitochondna is energy-dependent.

In agreement with predictions, d i s showed (Figure 16) that t h e was one band

present when the TnT r d o n product was assayed (precii~or APP), two main bands

after mitochondnal import @recursot plus mature APP), one main band when @ p i n

was added to the import reaction (manire, mitochondrial APP), and no bands in the

presence of valinomycïn. The import reaction also showed evidence of much smaiier

bands. These may be proteolytic products o f APP, suggesting that APP is processed

within mitochondria The resuits suggest that APP is targeted to and resides in

rnitochondria, where it may k fbrther prOcessed into smaiier species.

4.7.2 Dual color ih v b iluonsetnce microseopy of neuroblristoma ce1 linc

Chimeric APP-GFP pducts , expressed h m the pEGFP-NI plasmid (which

conîains no mitochondrial targeting sequence), were utilùed to v i s d k whether APP

transiently overexpressed in SK-N-SH newoblastoma cells is located within

mitochondria Chimeric protein coding regions were created by in-hmed fiision of APP

with GFP in the vector, and five such constnicts were designed as foilows:

1 pEGFP-N1 alone

. . 11 pCMVlmyclmitolGFP (COX VTiI mitochondrial cDNA, positive control

encodhg a mitochondrid targetiag sequence) (Rizzuto et al., 1992)

Üi P E G F P - N I ~ A P P ~ ~ ~ ~ ~ (ahost cornpiete coding region)

iv pEGFP-N I /APP ,,, ( N P , NHZ-termonai nucleotides 1 -208)

v mutant pEGFP-N l/APPmT (deletion mutam of APP, lacking nucieotides 1 -

mitos - œ + + + + trypsin - O - + + +

valinomycin - - O œ - +

Figure 16 Mitochondrial import in viîro. Lane 1. Translation pmduct of full-length APP, (Le. 35S-methionine-labe APP). Lane 2. Same as Lane 1, loading 1/5. Lane 3 '%-APP in presence of mitochondria. Lane 4. "S-APP in presence of mîtochonciria and trypsin, 500 pg/d. Lane 5. "S-APP in presence of mitochondria and uypsin, 250 pg/ml. Lane 6. "S-APP in presence of rnitochondna and trypsin (400 pg/ml). preceded by valinomy cin treatment .

1 I 1, i.e. the putative mitochondrial signai).

To isolate the APPmsmm, £iagment, ~ G E M ~ Z V A P P ~ ~ ~ ~ was restficted with

EcoR 1. For pGEMMAPP695m, there are three EcoR 1 sites, at bps - 15 of 5' UTR, plus

1795 and 2851 of the APP insert. Therefore, EcoR 1 -testrictai P G E M ~ ~ V A P P ~ ~ ~ ~

yielded three bands of 1 .O6 Kb, 1.8 1 Kb (= APP,,,,,), and 3.34 Kb (âata not shown)

The pEGFP-N 1 plasmid was amplXeci and verifid by EcoR 1 restriction. There

is one EcoR 1 site in the MCS of pEGFP-NI. Therefore, EcoR 1-restricted pEGFP-NI

yielded one band of 4.7 Kb (data not shown). Additional 2 Kb and 0.5 Kb supercoil

bands were seen. The manufacturer stated that additional supercoil bands were a normal

feature of the pEGFP-NI plasmid. To re-verify pEGFe-NI, two more restrictions of

pEGFP-N1 with EcoR 1 were carrieci out, the last restriction showing only the 4.7 Kb

band (data not shown).

Foliowing ligation of the APP,,,, fragment into EcoR 1-resiricted pEGFP-

N1 to produce the pEGFP-Nlf APP,,,, constnrt, an insert was verified by EcoR 1

restriction analysis. The pEGFP-NI/ APPemTlm5 consûuct has two EcoR 1 sites, at

each end of the APP insert. Therefore, EcoR I-restricted pEGFP-N1/APP695wT,,

yielded two bands of 1.8 Kb (APPa9YN-T1mS) and 4.7 Kb @EGFP-NI) (dafa not shown).

To verify the correct orientation of the insert, the coastruct was restricted with

B a d 1 . There are hvo BamH 1 sites in the pEGFP-N 1 /APPNNTI,, wnstruct, at bp 1329

of the APP Uisert, and in the MCS of pEGFP-NI, 3' to the APP insert. Therefore, BamH

1-restncteà, forrectly onenteà PEGFP-NI/APP~-~~,~ yielded two bands of 0.5 Kb and

6 Kb. BamH 1 -reshicted, inwrrectiy oriented pEGFP-N 1 /APPwT1,, yielded two bands

of 1.4 Kb and 5.2 Kb. Once agah supercol bands were present chat ovalapped the

expected bands, especially around the 0.4 Kb arwi (data not shown).

For fùrther verifidon of the conscnict, the DNA was cestricteci sepmtely with

EcoR 1 and BamH 1. In order to get a better sepration of the DNA bands, the s m d

AGE was repeated with 1.6% agarose Figure 17). The supercoi1 bands can now be

differentiated h m the expected bands.

The pEGFP-N I fAPP-- construct was prepared h m PEGFP-NI/APP~~, ,~

by restriction wïth Pst 1, followed by ligation of the linearized comtruct. EcoR 1

restriction d y s i s was used to ver@ the constructs, pEGFP-NlfAPP,,,. and

pEGFP-N 1. There is one EcoR 1 site in the MCS of each construct Therefore, EcoR 1 -

restricted pEGFP-N l/APPm5w- yielded one band of 4.9 Kb, and the EcoR 1 - d c t e d

pEGFP-NI yielded one band of 4.7 Kb (Figure 18). Expected bands are seen, plus the 0.5

Kb supercoi1 artifact.

To compare the fluorescence pattern created by the chimeric proteins with that

of the normal distribution of mitocbondria, the neuroblastomacells were aiso transfected

with a plasmid that expresses GFP fused to a mitochondriai targeting sequence,

pCMV/myc/mito/GFP. Followhg amplification of the pCMV/myc/mito/GFP plasmid,

it was verified by Pst 1 restriction analysis. There is one Pst 1 site in the MCS of

pCMV/myc/mito/GFP. Therefore, Pst 1 -restricted pCMV/myc/mito/GFP yielded one

band of 5.7 Kb (Figure 19).

SIC-N-SH neuroblastorna cells were tramiently transfected with the chimeric

DNA constructs and pCMV/myc/mito/GFP. To determine the normal fluorescence

Figure 17 EcoR 1 & BamH 1 restriction of pEGFP-N1fAPP6sfi-T,, ligation constnicts. Lanes A 1-3. Unrestricted pEGTP-Nl. Lanes B 1-3. EcoR1-restricted pEGFP- N I /APP6m,m5. Lam C 1. High molecular weight DNA marker (Biorad). Lmes C 2-3. BamH 1-restricted pEGFP-Nl/APP,,,,,,. DNA was aained with ethidium bromide in 1.6% agarose. Expected DNA bands can now be dinerentiated Erom supercoil artifacts.

Figure 1 8 EcoR 1 restriction of pEGFP-N 1 and pEGFP-N l/APP,,,,,,,. Lane 1. Unrestricted pEGFP-N i . Lane 2. EcoR 1 -restricted pEGFP-NI . Lanes 3 & 6.1 Kb DNA ladder (Gibw BRL). Lane 4. Unresaicted P E G F P - N I / A P P ~ ~ , ~ - ~ ~ ~ . Lane 5. EcoR 1 - restricted pEGFP-N1/APP695,. The expected 4.7 Kb (pEGFP-NI) and 4.9 Kb (pEGFP-N L/APP6,,,,) DNA bands are present. DNA stained with ethidium bromide in 0.8% agarose gel.

Figure 19 Pst 1 restriction of pCMV/mydmito/GFP. Lane 1 . Unrestricted pCMV/myc/mito/GFP. Laue 2. 1 Kb DNA ladder (Gibco BRL). Law 3. Pst 1 -restricted pCMV/myc/mito/GFP- Stained with ethidium bromide in 0.8% agarose gel.

pattern of mitochondria in aeuroblastoma ceus, and to create the dual color overlay

(green of GFP plus red of MitoTracket Red produces a yellow colour when overlayed),

the cells were ais0 treated with the with the rnitochondrial probe, MitoTracker Red.

Unifonn cytoplasmic and nuclear fluorescence was detected in pEGFP-N1

transfectants (Figure 20 a). pEGFP-Nl/mito transfêctmts fluoresced in a punctated

perinuclear pattern tbat CO-localized with Iilitochondrial fluorescence within the same celi

(Figure 20 b). Consistent with a mitochondrid location, pEGFP-NI/APP m5m-T,m

localized to cytoplasmic areas of punctated fluorescence (Figure 20 c). pEGFP-

N 1 /APP695/N-m fluorescence was identicai to that O bserved with pEGFP-N 1 /mita,

strongiy indicative of a mitochoncùial location (Figure 20 d). Mutation of the NH,-

temllnal domain of APP @EGFP-N I/APPwT) abolished mitochondrial fluorescence

(Figure 20 e). These redts indicate that the NH,-terminus of APP is capable of targeting

this protein to mitochondria These hdings have also been verifïed in human

teratocarcinomaderivedNT2 ceiis (Sûatagene, datanot show). However, the resolution

seen with this technique is not adequaîe to make a dennitive daim, and therefore, future

studies will involve the use of confocal mimscopy.

4.7.3 Immunalectron mkroreopy to detect endogenous APP and PAP in mammaüan tissue

To detennine whether endogenous AFP is fomd in mitochondria, nwmdian

Figure 20 SK-N-SH neuroblastoma celi mitochondrial labeiling. Cells were transientiy transfected with pCMV/myc/mito/GFP or pEGFP-NI or its APP fusion proteins. as weii as being stained by MitoTracker Red. The fkst coiumn uidicates transient transfections with a. pEGFP-N 1, b. pCMV/mydmitolGFP, c. pEGFP-N 1/APP6H,x-r,,,, d. pEGFP-N IfAPP,,,-, e. pEGFP-Nl/APP,,&+ in which pEGTP-N I and its fusion proteins appear green. The second column represents the same neuroblastoma cells stained with MitoTracker Red, which appear red. The third column represents the above red and green superùaposed images which, when green areas are directly superimposed on red images, produces a yeiiow colour, and indicates areas of the ceil where pEGFP-NI and its APP fùsion proteins have localized.

tissue sections were examined by immmoelectmn microscopy, ushg goldamjugated

antibodies to APP. immunelectron mimscopy analysis confirmed the presence of APP

associated with the mitochondrial crïstae, the site of COX 1 localization in human, pig

and rat brain tissues, plus human kidney tissues (Figure 21)-

4.7.4 Iniaunoekctmn uticmscopy to detect endoge~ous APP and P A P in AD and control braias

Since AD is associated with a reduction in COX activity as weil as over-

accumulation of amyloidogenic fragments, it is possible that mitochondrial occupation

by APP and PAP may be more marked in AD versus non-AD brains. To examine this,

immunoelectron microscopy was performed to detect endogenous APP and PAP in

brains of AD patients and non-AD controls. Results show that APP locaüzed to

mitochondria and other membrane-bomd organelies in both AD (Figure 22 a) and non-

AD sections (armws indicate mitochondria). Forty to fifty percent of mitochoadna

retallied at least three gold particles, and both membranous and lumenal regions of

mitochondria were seen to be labelled. However, there was no apparent diffêrence in

degree of APP labelling of mitochondria h m AD and non-AD specimens. PAP had a

similar distribution to that of APP, except that the fkquency of PAP in mitochondria of

AD sections (Figure 22 b) appeared to be higher than that of non-AD sections (Figure 22

c). This mvel nisding suggests, similar to the mitochondrial import assay resuits, &ai

APP may be processed in mitochondria to release amyloidogenic peptides.

Figure 21 Lodïzation of APP by immunoelectron microscopy in macnmalian brain and kidney. a. Human brain, b. Human Lidney, c. Pig brain, d. Rat brain. Visuaiization of APP was performed using mouse ami-human APP and goat, d-mouse gold- conjugated IgG (gold particle size, 10 am). Control sections were incubated with mouse anti-IgG. LabelLing of APP is seen clearty by gold particles associated with the rnitochondnal cristae (arrows). Scale bar: 1 cm = 5.0 p m in (a), 0.16 p m in @). 0.24 pm in (c), 3 -4 pm in (d). The images were enhancd in size 2.0-4.0 fold.

Figure 22 Localkation of APP and PAF by immunoelectron microscopy in human AD and non-AD brain. a. Human brah, APP, b. AD braip PAP. c. non-AD brain, PAP. Visualization of APP was perfomed using mouse ami-human APP and goat anti-mouse, gold-conjugated IgG (gold particle size, 10 nm). Control sections were incubated with mouse anti-IgG. Visualkation of PAP was perforrned ushg rabbit anti-P AP and goat anti- rabbit, gold conjugated IgG (gold particle sue 5 nm). Labelhg of APP is seen by gold particles associated with the mitochondria (arrows) and other membrane-bound organelles. Scale bar: the images were enhanced in size 2.0-4.0 fold.

The pathophysiology involved in AD is very complex, and incorporates an

integration of developmental and late life events, encompassing genetic and

environmental factors, It is clear several biochemicd processes are involved in various

converging pthways that leaà to the histopathology of AD. Of these, deficits in energy

metabolism appear to play a fiindamental d e .

Our resuits demoostrate, for the nrst tirne, that a protein of rnitochondnal ETC-

specific origin (COX I) (Davis et al., 1994) interacts with a cytoplesmic membrane-

bound protein (APP), aad that APP and PAP may directiy influence mitocbondrial energy

metabolism by their effects on COX. The ability of COX 1 to interact with APP appeam

to be distinct h m this protein's He-sustainhg mle in oxidatlve respiration (Zbang et al.,

1990). Bath APP and COX 1 are highly consemeci and widely expressed in eukaryotes

(Anderson et al,, 198 1 ; Tanzi et al., 1987; Yamada et al., 1989). The fzt that APP is

expressed in most cells and that the APP gene promoter is similar to that of housekeeping

genes (Salbaum et al., l988), suggests a fiindamental rde for this protein. For example,

the presence of APP and PAP in mitochondria of non-AD brain suggests a mle for these

proteins in normal mitochondrial hction. The APP-COX interaction may thus serve

another conserveci d e , which c d d pmvide several links between energy metaboüsm

with other aspects of AD, such as free radical and PAP generation that initiate neuronal

apoptosis. The dysregulatim of this mitochondriai-APP/W interaction may contribute

to the pathogenesis of AD. ECOX 1 binds speciflcally with APP, then overexpression

of APP may modulate the activity of COX 1, thereby indirectly inhiiiting COX, which

will lead to deficits in energy metabolism. This is consistent with the findings that

overexpression of APP in cultured human muscle fibres d t e d in a specifically

decreasd COX activity and severe mitochondrial ultrastnrtural abnorsalities (As-

et al., 1996). This mechanism may also be responsible for mitochondrial and COX

abnormalities in the brains of AD patients. There is evidence that there are deficits in

energy metabolism in the braias of patients with AD (Blass and G i n , 1991).

Although proteolytic cleavage ofAPP generates the PAP species thet are the main

component. of the NPs associated with nemtoxicity (LaFeria et of., 1999, the detailed

mechanism of the association between APP and neurodegeneration are not precisely

defined. The present study was initiated to i denw APP effector-binding proteins in the

hope of deriving possible fùnctions for APP. A yeast two-hybnd system assay showed

cytochrome c oxidase subunit 1, one of the caîaîytic subunits of Complex IV of the

mitochondrial ETC, interacted with various APP isoforms (APP,, LWP,~,, APP,), and

that this interaction involved the extracellular domain of APP. The observed interaction

between APP and COX 1 wuld have been non-specific, because COX causes m u e n t

f a l s e p o s i t i v e s i n y e a s t t w o - h y b r i d s y s t e m s ( S e e

www. fccc. edu/researc~~~bs.go~ernis/llu~i~ fdse. hi). As weil, APP and COX 1 may not

be in their native conformation, orbe correctly pst-translationaliy modifieci in yeast, and

hence do not mimic metabolism in mammaiian ceiis. In addition, there are no known

examples of mitochondrial-specific enzymes interacting with membrane-bound

receptor/proteins. However, due to the co~ec t ion of APP and COX to AD, it was

important to investigate this interaction f ider.

Due to the documented cases of kficits in energy metabolism and COX activity

in AD, the detennination of mechanisms by which a COX deficiency might develop in

humans is of considerable clinical sigdïcance. This involves ûyhg to define how APP

could affect mitochondnal structure and fhction in AD. We propose that increased

levels of APP in mitochondtia of AD brah may have a causal relationship to the

corresponding decreased COX activity (Le. overeqression of APP causes a seledve

deficit of COX activity in AD brains). Therefore, it was important to investigate the

relationship between APP accumulation and COX deficiency in mitochondria

For APP to interact with a f8cultative mitochondrial protein, we postulate thaî the

interaction M e s place in the mitochomiria, and that therefore, APP must be targeted to,

and localized in, this organelle. In support of this theory, cornputer data base analysis of

the APP leader peptide sequence suggested APP contains a possible mitochondrial

targeting sequena. Based on this analysis, we tested our hypothesis that APP may also

reside in mitochondria and under certain conditions, impair the activity of COX, and

subsequentiy, the ETC.

A markedly reduced level of total COX activity in the celis overexpressing

mitochondria-targeted APP was found d e n compareci with other traasfected constructs,

including those transfwted by a plasmid that caused generalized APP expression. In

con- no signüicant change was observed for cornplex I activity between the SK-N-

SH transfectants. It is notewoaby that nuclei-targeted APP or mitochondrïa-targeted a-1

antichymotrypsin (a PAP plaque-associated protein) overexpression did not significantly

affect COX activity in SIC-N-SH stable transfièctants. This suggests that the accumulation

of APP in mitochondria plays a crucial d e in specifïcally mediating decreased COX

activity.

Because mechanians of apoptosis that apply to neraodegeneration in g e n d may

also apply to AD (Smale et al., 1995), and have been tied to defects in energy

metabolism @cal, 1995; Greenluad et al., 1995), it was assessed whether APP-mediatecl

decreased COX activity potentiates apoptosis in SK-N-SH neumblastoma ceIl

transfectants. There was a prominent and signifiant rise in apoptosis only in those

neuroblastoma cells transfected with mitochondria-targeted APP, suggesting that

overexpression of APP on its own may not be sufncient to induce apoptosis in neurons

without the accumulation of APP in mitochondria

The excessive production of ROS that ultimately leads to initiation of apoptosis

has been suggested as the Link between a generalized defect in bioenergetics and the

pathogenesis of AD (Coyle and Puttfatken, 1993). To determine the role of ROS in APP-

mediated neuronal apoptosis, the levels of ROS were rneasured in neuroblastoma ceus

targeted with overexpression of APP. There was markedly increased ROS generation

only in those cells stably tmnsfected with mitochondria-targeted APP. Thus, it appears

the generation of ROS by APP overexpression depends on an APP-mitochondrial

association. Measuring Lpid peroxidation, it was found hat the i n c d free radical

generation resulted in cellular âamage in the neuroblastoma cell transfectaats. However,

the lipid peroxidation was only sigdicantly elevated in the APP-mitochondria-targeted

SK-N-SH transfeftants. These results suggest that a defect in metabolism of ROS in

mitochondria-targeted APP ovaexpressing SIC-WH celis leads to apoptosis, and that

mitochondria are intimately involveci in the prooess.

To assess the possibility that enhanced ROS production may be due to an APP-

mediated effect on ROS savenger enzymes, the activation of Eree radical scavenging

enzymes, glutathione pemxidase and supemxide dismutase, was asses&. APP had w

signincant effect on the activity of glutathione peroxidase (-01 et al., 1983) in the

stably transfected cells compared with mntrol cells, but there was a si@cant increase

in SOD activity (Nagi et al., 1995) in the APP-targeted traosfectants compared with other

transfectants and control ceus. Thus* only mitochondria-targeted APP once again had an

effect. This action may be a sign of a compensatory response by the enzyme to increased

ROS production.

For APP to interact with a mitochondrial m e , APP must be targeted to and

localized in mitochondtia. A classic in vitro mitochondrial import assay showed that an

apparent precursor, 35S-methionine-labeled APP, was imported into rat Liver

mitochondria in an energydependent manner, and that once in mitochondna it is cieaved

into a shorter mature form This assay seemed to also demonstrate the important finding

that APP may be fbrther processed in the mitochondna into shorter products.

Two APP constructs wntaining7 respectively, the NHZ-terminal 1795 and 208

base pairs, when ûansiently overexpRssed in SK-N-SH newoblastoma cells, created

fluorescence patterns consistent with a mitochonàrial location, while a mutant APP

construct with the nrst 11 1 NH,-- base pairs deleted, showed a pattern of

generalized ldization. These d t s indicate, for the nrst time, that APP is targeted to

mitochondria, and that the Mi, temiinus of APP is capable of, and necessary for, this .

targeting (Le. the NH, terminus of APP contains a mitocbondrial targeting sequence).

However, if this is the case, it is puzzling to me why in the previous experiments on

stably transfected cells, the APP errpressed h m the pCCneo/APP wostnrts did not also

adversely affect the parameters measured (at least, not significantly). Although

specdative, perhaps this is due to the different parameters king examined in the two

experimentai settùigs.

Because the resolution of the previous experiment was not sutncient to

conclusively wnfirm the presence of APP in mitochondria, elecbon microscopy was next

employed, and c o n f d mimscopy will be employed in the future. Immunoelec~n

microscopy analysis, utilizing gold-conjugated antiboâies, codbmed the presence of

APP associated with the mitochondrial cristrie, the site of COX localization, in human,

pig, and rat brain and in buman kidney. As AD is associated with a reduction in COX

activity as weii as over accumulation of amyloidogenic fhgments, it is possible that

mitochondnal occupation by APP and PAP may be more marked in AD versus non-AD

brains. To examine this, immunoelectmn rnicmscopy in both AD and non-AD sections

were performed. Forty to n f t y percent of mitochoadria retained at least three gold

particles, and both membranous and lumenal regions of mitochondria were seen to be

labelled. However, there was no apparent ciifference in de- of APP labelling of

mitochondria h m AD and non-AD specimens. PAP had a similar distribution to that of

APP, except that the fkquency of PAP in mitochoncîrïa of AD sections appeared to be

higher than tbat of non-AD sections, suggesting that, in AD, there may be enhanced

amyloidogenic processing of APP inside mitochoadna

Our studies thus support the hypothesis that APP is targeted to mitochondria,

probably the innet mitochondrial membrane, where its NH,-te~minal domain interacts

with COX 1. W e have also provided evidence for the link between APP metabolism and

dinetent features of AD, such as mitochondrial dysfiinction (deficit in COX activity),

oxidative stress (enhanced ROS levels and lipid peroxidation), and neuronal degeneration

(iïpid peroxidation and apoptosis)- The resuits are consistent with the hypothesis that

accumulation of APP in mitochondria could lead to aberrant APP-COX 1 interaction and,

consequently, decreased COX activity. This, in turn, causes the enhanced generation of

ROS levels and Iipid pemxidation, leading to neuronal apoptosis. The alteration in

energy metabolism may also alter the proçessing of APP and induce potentially

amyloidogenic peptides (Gabuzâa et al., 1994), which cause and perpetuate a cycle of

mitochondrial and cellular neuronal degeneration and apoptosis.

The mechanisms by which incomplete oxidation affects APP metabolism have

remaiwd unresolved. As suggested by our results, the direct interaction of COX 1 with

APP and hence the close pmximity of this interaction to the temiinal ETC (Hill, 1994),

may provide an immediate environment for the reaction of ROS with APP. This reaction

may lead to abnormal processing of APP or, dternatively, the radical-iaduced formation

of PAP. The interaction between COX I and APP could provide evidence of the direct

involvement of COX 1 activity specifically in the metabolism of APP.

Altematively, it could be argued that the accumulation of APP and PAP species

in the brains of AD patients may only reflect a generalized accumulation of amyloid

proteins in this condition. However, there was seen to be an apparent increase in

mitochoriclriai BAI? in sections h m AD compared to non-AD brains. This suggests that

there is e n b a n d amyloidogenic pmcessing of APP in the mitochondria of AD patients.

Other unresolved features of AD, such es neuronai apoptosis, may also be related

to the interaction between COX I and APP. This interaction may affect the release of

cytochrome c, which is also coupled to COX and thought to be critical in the cascade of

events leadhg to apoptosis (Liu et d., 119%; Kluck et al., 1997; Yang et al., 1997). It is

known that caspases cleave APP to release an apoptosis-related, amyloidogenic peptide

(Gervais et al., 1999; Weidemann et al., 1999), and that caspases are fcund in the

mitochondria (Susin et al., 1999, Krajewski et al., 1999). The inner mitochondnal

membrane may act as a site where the intra-membrane cleavage of APP occurs.

Therefore, accumulation of APP in mitochondria could lead to abnormal processing of

APP and increased generaîion of amyloidogenic peptides.

Although the function of APP in the brain is as yet unkwwn, an attractive

hypothesis for the biological role for the APP-COX interaction may be that APP is

involved in the translocation of COX 1. Aiternatively, because both APP,,, and APP,,,

contain a 56 amino acid segment encoding a Kunitz protease inhibitor domain (KPI)

(Kitaguchi et al., 1990; Van Nostrand et al., 1989) in addition to the APP, primary

sequeace, APP may be involved in the processing of non-assembled inner mitochondrial

membrane proteins.

The localization of APP and PAP to mitochondria is novel and warrants M e r

investigation into the hction of these proteins in mitochondria Several parsmeters

r e k mresolved. For example, whether the pethophysiology is altered by the

interaction of the different isoforms of APP and mutant APPs with COX 1, and

assessment of the bctional importance of this interaction in AD. Nevertheles, our

results have generated a molecular mode1 that will be usefirl in studying the regdation

of this interaction by neurologid fstors, and therapeutic dnigs tbat interfere with the

early stages of the disease.

in conclusion, we suggest that the events involved in the pathophysiology of AD

can be tightly associated with deficits in energy metabolism through the interaction

between APP and COX 1.

6.0 REFERENCES

Anderson S, Bankier AT, Barrell BG, de Bruijn MH, Coulson AR, Drouin J, Eperon IC, Nierlich DP, Roe BA, Sanger F, Schreier PH, Smith AJ, Staden R, Yomg IGAnderson S, et (198 1) Sepuence and orgMization of the human mitochondrid genome. Nahrre; 290(5806): 457-65.

Anderson JP, Esch FJ, Keim PS, Sambamurti K, Liebergurg 1, and Robakis NK, (1991) Exact cleavage site of Aizheîmer amyloid preciirsor in neuronal PC-12 ceiis. Neurosci Lett.; 12%: 12642%.

Anderson AJ, Pike CJ, and Cotmsui CW. (1995) Differential induction of immediate eariy gene pioteuis m CUItured neumns by beta-amyioid (A beta): d a t i o n of c-Jun with A beta-induced apoptosis. 1 Neurochen ; 65: 148701498.

Araujo D and Cotman C. (1992) Beta-emyloid stimulaies glial cells in vitro to pmduce growth fxtors that accumulate in d e plaques in Aizheimer's disease. Bruin Res. ; 569: 141-145.

Arispe N, Rojas J, and Pollad HB. (1993) Alzheimer disase amyloid P protein forms calcium channels in bilayer membranes: blockade by tromethamhe and aluminium. Proc, Nàî- Acad Sei.; 90: 567-571.

Askanas V, M c F h J, Baque S, Alvsra RB, Sarkozi E, and Engel WK. (19%) transfer of P-amyloid precursor pmetein gene using adewvirus vector causes mitochondrial abnormalities in cultured normal human muscle. Proç. Nat- Acad Scf; 93: 1314-1319.

Barnes NY, Li L, Yoshikawa K, Schwartz LM, Oppenheim RW, and Milligan CE. (1 998) hcreased production of amyloid precursor protein provides a substrate for caspase-3 in dying motomeutons, J. Netuosci.; 18: 5869-5880.

Beal MF. (1 992) Does impairment of energy metabolism result in excitotoxic neuronal death in neurodegenerative illnesses? A m Neurol. ; 3 1 : 1 19-1 30.

Beal MF. (1995) Aging, energy, and oxidative stress in neumdegenerative diseases. Ana Neurol; 38: 357-366.

Beal MF. (1997) New techniques for investigaihg mitochondrial DNA in neurodegenerative diseases. Nwology; 49: 907-908-

Behl C, Davis J, Cole GM, and Schubert D. (1992) Vitamin E protects nerve celis h m amyloid P protein toxicity. Bichem. Biophys. Res. Commun; 186: 944-952.

Behl C, Davis JB, k l e y R, and Schubea D. (1994 a) Hydrogen peroxïde mediates amyloid &protein toxicity, Ce& 77: 8 17-827.

Behi C, Davis JB, Kiier FGy and Schubert D. (1994 b) Amyloid B protein induces necrosis rather than apoptosis. Bran Res.; 645: 253-264.

Bennett MC, Diamond DM, Sûyker SC, Parks JK, and Parker W. (1992) Cytochrome oxidase inhibition: a novel animai mode1 of Alzheimer's disease. J- Gen'atr- Psychiatry Neurol; 5 ; 93-1 01,

Benveniste H, Dreyer J, Schousboe A, and Diemer NH. (1984) Elevation of the extracelida. concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral mictûdialysis. J. Neurochem. ; 43 : 1 369- 1 374.

Bemi G and Moretti k(1995) Are reactive oxygen species involved in Alzheimer's disease? NewobioL Aging; 16: 66 1-674.

BIass JP, Sheu RK-F, and Cedarbaum M. (1 988) Energy metaboüsm in disorders of the nervous system. Rev. N e d ; 144: 534-563.

Blass JP and Gibson GE. (1991) The role of oxidative a b n o d t i e s in the pathophysiology of Alzheimer's disease. Rev. Neurd. ; 147: 5 13-525.

Breen KC, Bruce M, and Anderton BH. (1 991) &ta amyloid precursor protein mediates neuronal celi-cell and celi-surface adhesion. Neurosci. Res. ; 28(1): 90- 1 00.

Brustovetsky N and Küngenberg M. (1996) Mitochondrial ADPIATP d e r can be reversibly conveited into a large channel by CS+. Biochemishy; 35(26): 8483-8488.

Busciglio J, Gabuzda DH, Matsudaira P, and Yankner BA. (1993) Generaîion of beta-amyloid in the secretory pathway in neuro~litl and nonnemaal ceiis. Proe. Nat. Acad Sci-; 90(5): 2092-2096.

Busciglio J and Yanker B (1995) Apoptosis and increased genecation of reactive oxygen species in Down's syndrome n e m m in vitro. Nume; 378:776-779.

Caporaso GL, Gandy SE, Buxbaum ID, and Greengard P. ( l m ) Chlomquine inhibits intracellular degradation but not secretion of Alzheimer betdA4 amyloid precursor protein. froc. Nat- A c d W.; 89(6): 2252-2256.

Camagnol F, Sinet PM, and Jerome H. (1983) Seleniumdependeat and non- seleniumdependent glutathione petoxidases in human tissue extractS. Biochimicu et Biophysia Acta; 759: 49-57.

Casari G, De Fusco M, Ciamatori S, Zeviani M, Mora M, Ferandez P, De Michele G, Filla A, Cocozza S, Marconi R, M A , Fontaine B, and Ballabio A. (1998) S@c paraplegia and OXPHOS impairment caused by muiations in paraplegia, a nuclear-encoded rnitochondrial metallopmtease. Cell; 93(6): 973-983.

Chaodrasekaraa K, Giordano T, Brady DR, Stoii J, Martin U, and Rapoprt SI. (1994) Impairment in mitochondrial cytochrome oxidase gene expression in Alzheimer disease. Bruin Res, Mol. Br& Res.; 24(1-4): 336-40 .

Chandrasekaran K, Hatanpaa K, Brady DR, Stol1 J, and Rapport SI. (1998) Downreguiation ofoxidative phosphorylation in Alzheimer disease: los of cytochrome c oxidase subunit mRNA in the hippocampus and entorhinaI cortex. Bruin Res. ; 7%( 1 -2): 1 3 - 1 9.

Chandrasekaran K, Hatanpaa K, Brady DR, and Rapoport SI. (1996) Evidence for physiological dom-regulation of brain oxidative phosphorylation in Alzheimer's disease. fip- NeuroI-; l42(l): 80-88.

Chandraselorrao K, Hatanpaa K, Rapport SI, and Brady D R (1997) Decreased expression of nuclear and mitochondrial DNA-encoded genes of oxidative phosphorylation in association neococtex in Alzheimer disease. Brain Res. Moi- Brain Res. ; 44(1): 99-104.

Chartier-Harlin MC, Crawford F, Houlden H, Warren A, Hughs D, Fidani L, G a t e A, Rossor M, Roques P, Hardy J, and Muilan M. (1991) Early-onset Alzheimer's disease caused by mutations at codon 717 of the p-amyloid p r e c m r protein gene. Nature; 3 53 : 844-846.

Chow N, Korenberg IR, Chen XN, and Neve RL. (19%) APP-BPI, a novel protein that binds to the carôoxy-terminal region of the amyloid precursor protein. J. Biol. Chem.; 271(19): 11339-1 1346.

Christie RH, Freeman M, and Hyman BT. (19%) Expression of the macrophage scavenger receptor, a multifimctionai iïpopmtein receptor, in mimglia associateci with senile plaques in Aizheimer's disease. Am- J. PathoL; 148(2): 399-403

Cohen ML, Golde TE, Usiak MF, Younkin LH, and Younkin SG. (1988) In situ hybridization of nucleus basalis newons shows increased beta-amyloid mRNA ia Alzheimer disease. Proc. Nat. A c d Sci. ; 85(4): 1 227- 1 23 1.

Cordell B. (1994) beta-Amyloid formation as a potential therapeutic m e t for Alzheimer's disease. A n m Rev. Pharmacol. Toxicoll.; 34:69-89.

Corder EH, Saunders AM, Strittmatter WJ, et al. (1 993) Gaie dose of apolipoprotein E type 4 ailele and the increased risk of Alzheimer's disease in late-onset families. Science; 26 1 : 92 1-923.

Co- CW and Su IH. (1 9%) Mechanisms of neuronal death in Alzheimer's disase. Brain Pathol. ; 6: 493-506-

Cotrnan C and Anderson A. (1 995) A potential role for apoptosis in neurodegeneration and Alzheimer's disease. Mol, Neurobiol.; 10: 19-45.

Coyle JT and Puttfarcken P. (1993) Oxidative stress, glutamate, and neurodegenerative disorders. Science; 262: 689695.

Cummings JL, Vinters H V , Cole GM, and Khachaturian ZS. (1998) AldKMer's disease: Etiologies, pathophysiology, cognitive reserve, and m e n t opportunities. Neurology; 5 1 : S2-S 17.

Curti D, Rognoni F, Ga,"parini L, Cattaneo A, Paoli110 M, k h i M, Zani L, Bianchetti A, Trabucchi M, Bergamaschi S, and Govoni S. (1997) Oudative metabolism in cultured fibroblasts denved h m sporadic Alzheimer's disease (AD) patients. Newosci Left. ; 236( 1): 13-6.

Dank. D. (1983) Hereditary disorders of copper metabolism in Wilson's disease and Menke's disease, in m e Metabdic Basis of Inherited Disease, edition 5, Stanbuy JB, Wyngaarden JB, Fredticksen DS, Golstein JL, Brown MS, eds. (McGtaw-Hill New York), pp. 126 1 - 1266.

Davis P W Jr., Parks J, FiUey CM, and Kleioschmidt-De Masters BK. (1994) Electron transport chah defeçts in Alzheimer's disease brain. Newol. ; 44: 1090-10%.

Davis J, McMuwy H, and Schubert D. (1 992) The amyloid beta-protein of Aizheimeis di- is chernotactic for mononuclear phagocytes. Biochern. Biop&ss Res. Com~z(n. ; 1 8!?(S): 1096-1 100.

De Stmoper B, Umans L, Van Leuven F, and Van Den Berghe H. (1993) Study of the synthesis and secretion of normal and artificial mutants of murine amyloid precursor protein (APP): cleavage of APP occurs ina late cornpartment of the default secretion pathway. J. Cell biol. ; 12 l(2): 295-304.

Devereux J, Haeberli P, and Srnithines 0. (1984) A comprehensive set of sequence analysis pmgrams for VAX. Nucleic Acid Res. ; 12: 387-395.

Dewji NN and Singer SJ. (19%) Specific tramcellular bindiog baween membrane proteins aucial to Alzheimer's disease. Proc Nat. Acad Sfî ; 93 : 1257542580.

Dewj*i NN and Singer SJ. (1997) The seven-trammembrane-spanniog topography of the Alzheimer disease-related pcesenilin proteins in the plasma membranes of cultured cells. Proc Nat. Acad Ski. ; 94: l4îOîS- 14030.

h u i s A, Sebben M, Haynes H, Pin JP, and Bockaert J. (1988) NMDA reçeptors activate the arachidonic acid cascade system in Stnatal nemns. Naiwe; 336: 68-78.

Wks T, Dy* E Hattmann T, Masters C, and Beyreuther K. (1 992) Amyloidogenicity of PA4 and PA4-b&g amyloid protein precursor fhgments by metalcatalysecl oxidation. J. Biol. Chem.; 267: 1û21û-18217.

Dyrks T, Weidemann A, Multhaup G, Salbaum JM, Lemaire HG, Kang J, Muller-Hill B, Masters CL, and Bayreuther K. (1988) Identification, trammembrane orientation and biogenesis of the amyloid A4 precursor of Alzheimer's di-. EMBO A ; 7(4): 949-957.

Earnshaw WC. (1999) Apoptosis. A cellular poison cupboard. Naiwe; 397: 387-389.

Wand SD, Silverman IM, Peskiad ER, Tsuang D, Wijsman E, and Moms JC. (19%) I n c d risk of dementia in mothers of Alzheimer's disease cases. Newofogy; 47: 254-256.

El Khoury J, Hickman SE, Thomas CA, Cao L, Silverstein SC, and Loike JD. (1996) Scavenger receptor-mediated achesion of mimglia to B-arnyloid fibrils. Nature; 382: 716-719.

Esch FS, Keim PL, Beattie EC, Blacher RW, Culwell AR, Oltersdorf T, McClm D, and Ward PJ. (1990) Cleavage of amyloid beta peptide during constitutive ptocesshg of its precursor- Science; 248: 1 122-1 124.

Estus S, Golde TE, Kunishita T, Blades D, Lowery D, Eisen M, Usiak M, Qu X, Tabira T, Greenberg BD, and Younkin SG. (1992) Potentially amyloidogenic, carboxyl-terminal derivatives of the amyloid protein precutsor. Rience; 255: 726 728.

Forloni G, Bugiani O, TagliaWii F, and Salmona M. (1996) Apoptosis-mediated neurotoxicity induced by beta-amyloid and PrP firagments. Mol. Chem. Neuropathol.; 28: 163-171.

Forloni G, Chiesa R, SmVoldo S, Verga L, Salmona M, Tagliavini F, and Angeretti N. (1 993) Apoptosis mediated neurotoxicity induced by chronic application of beta

amyloid fiagrnent 25-35. Necrro Reporl; 4: 523-526.

FuUer SJ, Storey E, Li QX, Smith Ai, Beyreuther K, and Masters CL. (1995) Intxacellular production of BA4 amyloid of Alzheimer's disease: modulation by phosphoramidon and lack of coupüng to the secretion of the amyloid precursor protein. Biochem. ; 34: 809 1-8098.

Furukawa K, Barger SW, Blalock EM, and Mattson MP. (19%) Activation of K+ channels and suppression of neurod activity by secreted P-amyloid precursorprotein. Nature; 379: 74-78.

Gabuzda D, Birriglio I, Chen LB, Matsudaira P, and Yankner BA. (1994) Inhibition of energy metabolism alters the ptocessing of amyloid precursor protein and induces a potentiaüy amyloidogenic derivative. A BioL Ckm; 26qf 8): 13623-1 3628.

Gabuzda Dy Busciglio J, and YanhKi BA. (1993) Generation of a potential precursor of beta amyloid h m immature APP in the secretory pathway. Suc. Neurosci. Absh. ; 19: 19.

Games D, Adams D, Aiessandrini R, Barbour R, Berthelette P, Blackweil C, Carr T, Clemens J, Donaidson T, Gillespie F, et al. (1 995) Alzheimer-type neuropathology in transgenic mice overexpressing V7 17F beta-amyloid precursor protein, Nature; 373 : 523-527.

Gasparini L, Racchi M, Benussi L, Curti D, Binetti G, Bianchetti A, Trabucchi M, and Govoni S. (1997) Effect of energy shortage and oxidative stress on amyloid ptecursor protein metabolism in COS ceils. Neurosci, Lett. ; 23 l(2): 1 1 3- 1 1 7-

Gave1 Y and von Heijw G. (1990) Cleavage-site motifs in mitochondrïal targethg peptides. Protein Eng. ; 4( 1): 33-37.

Gervais FG, Xu Dy Robertson GS, Vaillancourt JP, Zhu Y, et al. (1999) Involvement of caspases in proteolytic cleavage of Alzheimer's amyloid-p precursor protein and amyloidogenic AP peptide formation. Cell; 97: 3 9 5 4 6 .

Gibson GE, Sheu KF, and Blass Ji? (1998) Abnodi t ies of mitochondrial enzymes in Alzheimer disease. J NèwaI. T r a m . ; 105(8-9): 855-70.

Giulivi C, Poderoso JJ, and Boveris A. (1998) Production of nitric oxide by mitochondria. J Biol. C k m ; 273(!8): 1 1038-1 1043.

Glenner GG and Wong C W. (1984 a) Alzheimef s disease and Dom's syndrome: sharing of a unique cerebrovascular amyloid fibril protein. Biochem, Biophys. Res. Commun; 122(3): 1 131-1 135.

Glenner GG and Wong CW, (1984 b) Alzheimer's disease: initial report of the purification and characterizaiion of a novel cerebrovascular amyloid protein. Biockm, Biop#ys. Res. Commun; 120(3): 885-890.

GLick BS. (1992) Protein import into isolated yeast mitochondria Methods in Cell BioL ; 34: 389-399,

Glick BS, Brandt A, Cunningham K, Muller S, Hallberg RL, and Schaîz G. (1992) Cytochmmes c, and 4 are sorted to the intermembrane space of yeast mitochondria by a stoptransfer mechanism. Cell; 69: 809-822.

Goate A, Chartier-Hariin MC, Md80 M, Brown J, C d o r d F, Fidani L, GiuHra L, Haynes A, king N, James L, Mant R, Newton P, Rooke K, Roques P, Talbot C, Pericak-Vance M, Roses A, Wiamson R, Rossor M, Owen M, and Hardy& (1 991) Segregation of a missense mutation in the amyloid precursor protein with familial Alzheimer's disease. Nature; 349: 704-706.

Goedert M. (1993) Tau protein and the nemfibdary pathology of Alzheimer's disease. T r e d Netuosci.; 1 6( 1 1): 460-465.

Golde TE, Estus S, Younkin LH, Selkoe DJ, and Younkin SG. (1 992) Processing of the amyloid protein p r e c m r to potentiaiiy amyloidogenic derivatives. Science; 255: 728-730.

Gomez-Isla T, Hofister R, West H, et al. (1997) Neuronal loss correlates with but exceeds neurofibriiiary tangles in Alzheimer's disease. Ann Neurol. ; 4 1 : 1 7-24.

GomeAsla T, West HL, Rebeck GW, et al. (1 996) Chica l and pathological correlates of apolipoprotein E €4 in Alzheimer's disease. A m Newoi.; 39: 62-70.

G d PF, Werner P, Hsu A, Olanow CW, and Perl DP. (19%) Evidence of neuronal oxidative damage in Alzheimer's disease. Am J. PathoI. ; i49(l): 2 1-28.

Govoni S, Bergatnaschi S, Gasparini L, et al. (1996) Fibroblasts of patients affkted by Down's syndrome oversecrete amyloid precuLsor protein and are hyporesponsive to protein kinase c stimulation. Neuro20g-y; 47: 1069-1 075.

Green DR and R d JC. (1 998) Mit~hondria and apoptosis. Skience; 28 1 : 1 309- 1 3 1 2.

Greenlmd US, Deckwerth TL, and Johnson EM. (1995) Superoxide dismutase delays neuronal apoptosis: a mle for reactive oxygen species in programxned ceIl deah. Neuron;l4: 303-33 1.

Guo Q, Fu W, Luo H, Sells SF, Geddes JIU, Bondada V, Rmgnekar VM, and Mamon MP. (1998) Par4 is a mediator of neuronal degeneration associated with the pathogenesis of Alzheimer disease. N a Med; 4(8): 957-962.

Haass C and Sekoe DJ. (1993) Cellular processing of beta-amyloid precutsor protein and the genesis of amyloid beta-peptide. Cell; 75: 1039-1042.

Haass C, Hung AY, Schlossmacher MG, Teplow DB, and Seikoe DJ. (1993) beta-Amyloid peptide and a 3-kDa fiagrnent are derived by distinct cellular mechanisms. J. BioL Chem ; 268(5): 302 1-3024.

Halliweil B and Gutteridge JMC, (1989) in Free Radicals in Biology and Medicine (Clarendon press, Oxford), edition 2, pp 1-8 1.

Hardy J. (1997) Amyloid, îhe presenilina , and Alzheimer's disease. Trends netuosci-; 20: 154- 159.

Hardy JA and Higgins GA. (1992) Alzheimer's disease: The amyloid cascade hypothesis. Science; 256: 1 84- 185.

Harman D. (1995) Free radical theory of aging: Alzheimer's disease pathogenesis. Age; 18: 97-1 19.

Hartmann T, Bieger SC, BNhl B, Tienari PJ, Ida N, AUsop D, Roberts GW, Masters CL, Dotti CG, Unsicker K, and Beyreuther K. (1997) Distinct sites of intracellular production for Alzheimer's disease A beta40142 amyloid peptides. Nature Med ; 3: 1016-1020.

Haming HP, Kiadetzky RG, Melnik B, Hemerici M, et aL (1986) Stimulation of the scavenger receptor on monocytes-macrophages evokes release of arachidonic acid metabolites and reâuced oxygen species. Lab. Imest. ; 55: 209-2 16.

Hensley K, Carney JM, Mattson MF, Aksenova M, Harris M, Wu JF, Floyd RA, and Butterfield DA. (1994) A mode1 for P-amyloid aggregation and neurotoxicity based on free radical g e n d o n by the peptide: relevance to Alzheimer disease. Proc. Nat. A d . Sci.; 91 : 3270-3274.

Higgans GA, Lewis DA, Bahmanyar S, Goldgaber D, Gajdusek DC, Young WG, Momson JI-& and Wilson MC. (1988) Differential regdation of arnyloid-g-protein mRNA expression within hippocampal neuronal subpopulations in Alzheimer disease. froc. Nd. Acad Ski.; 85: 1297- 130 1.

W BC. (1994) Modeliag the sequence of electron t r d e r reactions in the single turnover of reduced mammalian cytochrome c oxidase with oxygem J. B i d

Chem ; 269: 2419-2425,

Hockenbery DM, Oltval ZN, Y'm X, Milliman CL, and Korsmeyer SJ. (1993) kl-2 fiuictions in an antioxidant pathwy to prevent apoptosis. Cell; 75 : 24 1-25 1.

HoweU N, Kubacka 1, Xu M, and McCuliough DA. (1991) Leber's hereditaq optic neuropathy: involvement of the mitochondrial ND 1 gene and evidence for an intragenic suppressor mutation. Am J. Hum Genet.; 48: 935-942.

Hoyer S. (1993) Brain oxidative energy and relateci metabolism, neuronal stress, and Alzheimer's disease: A speculaîive hypothesis. J. Geriatric Psychatry- Neurol. ; 6: 3-13.

Huber S, Martin JR, Loflier J, and Moteau K. (1993) Involvement ofamyloid precusor protein in memory formation in the rat: an indirect antibody appmach. Brain Res.; 603: 348-352.

H d e y SM. (1 998) Neurodegeneration. Science; 282: 107 1.

Hyman BT. (1992) Down syndrome and Alzheimer disease. Prog. C h Biol. Res. ; 373: 123-142.

n<au T, Trapp BD, Song KS, Schlegd A, Lisanti MP, and Okamoto T. (1998) Caveolae, plasma membrane microdomains for alpha-~ecrefa~e-mediated pcessing of the amyloid precursor protein. J Biol. Chem.; 273: 10485-10495.

Ishü K., Sasaki M, Kitagaki H, Yamaji S, Sakamoto S, Matsuda K., and Mori E. (1997) Reduction of cerebellar giucose metabolism in advanced Alzheimer's disease. J. Nucl. Med ; 3 8(6): 925-928.

Itagaki T, Itoh Y, Sugai Y, Suemaisu N, Ohtomo E, and Yamada M. (1996) Glucose metaboiism and Alzheimer's dementia Nippon Ronen I g a h i riZaîshi, Abstr. ; 33(8): 569-72.

Iverfeldt K, Walaas SI, and Greengard P. (1993) Altered processing of Alzheimer amyloid precursor protein in response to neuronal degeneration. Proc. Na. Acad Sei-; 90: 4146-41 50.

Iwatsubo T, Odaka A, Suzuki N, M h w a H, Nukina N, and ihara Y. (1994) Visualization of A beta 42(43) and A beta 40 in senile plaques with end-specific A beta monoclonals: evidence that an initially deposited spefies is A beta 42(43). Neuron; 13: 45-53.

Jarrett JT, Berger EP, and Lansbury PT (1 993) The carboxy terminus of the beta amyloid protein is critical for the seeding of amyloid formation: implications for the pathogenesis of Alzheimer's disease. Biochemis~, 32(18): 493-4697.

J-tt JT and Lansbury PT. (1993) Seeding "one-dimensional crystallization" of amyloid: a pathogenic mechanism in Alzheimer's disease and scrapie? Cell; 73:1055-1058.

Jin LW, Ninomia H, Roch JM, Schubert D, Masliah E, Otem DAC, and Saito T. (1994) Peptides containing the RERMS sequence of amyloid betalA4 pmtein precursor bind ce11 surfsce and pmmote neurite extension. J. N e i a d . ; 14(9): 5461 -5470.

Johnson SA, McNeill T, Cordell B, and Finch CE. ( l m ) Relation of neuronal APP-75 l/APPd% mRNA ratio and nemitic plaque deasity in Ahkirnefs di-. Science; 248: 854-856.

Kang J, Lemaire HG, Unterbeck A, Salbaum JM, Masters CL, Gneschik KH, Multhaup G, Beyreuther K, and Muiler-Hill B. (1987) The preculsor of Alzheimer's disease amyloid A4 protein resembles a cd-surf" receptor. Nature; 325: 733-736.

Katchman A and Hershkowitz N. (1993) Early anoxia-induced vesicular glutamate release results h m r n o b ~ o n of calcium h m intracellular stores. J. Neurophysiol. ; 70(1): 1 -7.

Katzman R (1 986) Alzheimer's disease. N. Eng. J Med ; 3 14: 964-973.

Keller IN, Pang Z, Geddes JW, Begley JG, Germeyer A, Waeg G, and Mattson MP. (1997) Impaùment of glucose and glutamate transport and induction of mitochondrial oxidative stress and dysfùnction in synaptosomes by amyloid beta- peptide: role of lipid peroxidation product 4-hydroxynoned. J. Neu~ochem.; 69(1): 273-284.

Keiler IN, Pang 2, Geddes JW, Begley JG, Genneyer A, Waeg G, and Maîtson W. (1997) Impairment of glucose and glutamate transport and induction of mitochondrial oxidative stress and dysfimction in synaptosomes by amyloid beta- peptide: role of lipid peroxidation product 4-hydroxynonenal. L Newochem.; 69(1): 273-284.

Khachaturian ZS. (1994) Calcium hypothesis of Alzheimer's disease and brain aging. Ann N. Y. A c d Sei.; 747: 1-1 1.

Kish SJ, Bergeron C, Rajput A, Donc S, Mastrogiammo F, Chang LW, Wilson IM, DiStefaw LM, and Nobrega JN. (1992) Brain cytochrome oxidase in Alzheimer's disease. J Netuochem ; 59: 1 13-1 14.

Kish SJ, Bergeron C, Rajput A, DoPc S, Mastrogiacomo F, Chang U, Wilson JM, DiStefano LM, and Nobrega JN. (1995) Brain cytochrome oxidase in Alzheimer's disease. Neurochem,; 59(2): 776-9.

Kitaguchi N, TakahaShi Y, Tokushima Y, Shiojin S, and Ito H. (1988) Novel precuRor of Alzheimer's disease amyloid protein shows protease inhibitory activity. Nec; 33 1 : 530-532.

Kitaguchi N, Takahashi Y, Oishi K, Sbiojiri S, Tokushima Y, Utsunomiya T, and Ito H. (1990) Enzyme specincity of pmteinase inhibitor region in amyloid preciusor protein of Aizheimer.~ disease: D i n i t properties compared with protease nexin 1. Biochem. Biophys. Acta; 1038: 105-1 13.

Klegen-s A, Waker D, and McGeer P. (1994) Activation of macrophages by Alzheimer beta amyloid peptide. Biochem Biophys. Res. Commun ; l99(2): 984-99 1 .

Kluck RM, Bossy-Wetzel E, Green DR, and Newmeyer DD. (1997) The release of cytochrome c h m mitochondria: a prirnary site for Bcl-2 regdation of apoptosis. Science; 275: 1 132-1 136.

Knauer MF, Soreghan B, Burdick D, Kosmoski J, and Glabe CG. (1992) Intraceiiular accumulation and resistance to degradation of the Alzheimer amyloid A41 protein. Proc. Nd. Acad Sci- ; 89: 7437-7441.

Knops J, Lieberbutg I, and Sinba S . (1992) Evidence for a nonsecretory, acidic degradation pathway for amyloid precursor protein in 293 ceiis. Identification of a novel, 22-kDa, beta-peptide-containing intermediate. J. Biol. Chen ; 267: 16022- 16024.

Koh J, Yang LL, and Cotrnan CW. (1990) Beta-amyloid protein increases the VULflerability of cultured wdcal nemm to excitotoxic damage. Bmin Res.; 533: 3 15-320-

Koo EH, Park L, and Selkoe DJ. (1993) Amyloid beta-protein as a substrate interacts with extracellular matrix to promote neurite outgrowth. Proc. Nat. Acod ScL ; 90: 47484752.

Kosik K. (1994) The Alzheimer's disease sphinx: a riddie with plaques aud tangies. J. Cell Biol. ; 127: 1 50 1 - 1 504.

Kosik KS. (1 999) A aotable cleavage: Widing up with P-amyloid. Proc. Nat. A c d Sci,; 96: 25742576.

ffijewski S, Krajewska M, Ellerby LM, Welsh K, Xie 2, Deveraux QL, Salvesen GS, Bredesen DE, Rosenthai RE, Fiskum Gy and Reed JC. (1999) Release of caspase-9 h m mitochondria during neuronal apoptosis and cerebral ischemia hoc . NafL Acad Ski; %(IO): 5752-5757.

Kuentzef SL, tUi SM, Al- RA, Greenberg BD, and Raub TJ. (1993) The Alziieimer ka-amyloid protein prec~l~~r/pmtease nexin-Ii is cleaved by sxmtasz in a trans-Golgi secretory cornpartment in human neuroglioma celis. Biochem. J.; 295 (Pt. 2): 367-378.

LaFerla FM, Troncoso JC, Strickland DK, Kawas CH, and Jay G. (1997) Neurod ceU death in Alzheimer's disease correlates with apoE upWe aud intracellular A stabilization. J. Clin. Iiest . ; 100(2): 3 10-320.

La Feria FM, Tinkle BT, Bieberich CJ, Haudenschild CC, and Gilbert J. (1 995) The Aizheimerys A beta-peptide induces wurodegeneration aud apoptotic ceIl death in transgenic mice. Ndiae Genet.; 9: 2 1-30.

Mon-CazaI M, Pietri S, Cuicasi M, and Bockaert L (1993) NMDA-dependent superoxide production and neurotoxicity. Nuitue; 364: 535-537.

Lakis J, Glasco S, Miller SW, Thal LJ, and Davis RE. (1995) Production of reactive oxygen species correlates with decreased cytochrome oxidase activity in Alzheimer's disease cybrids. Soc. Newosci Abst; 2 1 : 979.

Lamb BT. (1997) Presenilins, amyioid-p and Alzheimer's disease. Nafzue Medicine; 3(1): 28-29.

Lansbury PT. (1997) Structural wurology: are seeds at the mot of neuronai degeneration? Neuron; 19(6): 1 15 1-1 1 54.

Lassmann H, Bancher C, Breitschopf H, Wegiel J, Bobinski M, Jellinger K, and Wisniewski KM. (1995) Cell death in Alzheimer's disease evaluated by DNA fragmentation in sihi. Acta NeuropothoL ; 89(1): 3 5 4 1.

Lazararewicz JW, Wroblewsk. JT, Palmer ME, and Costa E. (1 988) Neurophmnurcoloogy; 27: 765.

LeBlanc A (1995) Increased production of a 4 kDa amyloid beta-peptide in senun deprived human primary neumn cultures: possible involvement of apoptosis. J Neurosci, ; 1 5: 783 7-7846.

LeBlanc AC, Kovacs DM, Chen HY, Villare F, Tykocinski M, Autilio-Gambetti L, and Gambetti P. (1 992). Role of amyloid precu~sor protein (APP): study with antisense

transfection of human neuroblastoma cells. J. Neutosci. Res. ; 3 1 : 635-645.

Ledesna M, Bonay P, Colaco C, a d Anla J. (1994) Analysis of microtubule-associated protein tau glycation in paireci helical filaments. J. Biol. Chem. ; 269: 2 16 14-2 16 19.

Lemere CA, Lopera F, Kosik KS, Lendon CL, Ossa J, Saîdo TC, Yamaguchi H, Ruiz A, Martinez A, Madrigal L, Hincapie L, Arango JC, Anthony DC, Koo EH, Goate AM, Selkoe DJ, and Arango JC- (1996) The E280A presenilin 1 Alzheimer mutation produces i n c d A beta 42 deposition and severe cerekllar pathology. Nature Med; 2(10): 1146-1 150.

Lendon CL, Ashall F, and Goate AM. (1 997) Exploring the etiology of Alzheimer's disease using molecular genetics. JAUQ; 277: 825-836.

Levy-Lahard E, Wasco W, PoorLaj P, Romano DM, Oshima J, Pettingell WH, Yu CE, Jondro PD, Schmidt SD, Wang K. et al. (1995 a) Candidate gene for the chromosome 1 familial Alzheimer's disease locus. Science; 269: 973-977.

Levy-Lahard E, Wijsman EM, Nemens E, Andertson L, Goddard KA, Weber JL, Bird TD, and Schellenberg GD. (1995 b) A faniilid Alzheimer's disease locus on chromosome 1 - Science; 269: 970-973,

Li WP, Chan W, Lai W, and Yew DT. (1997) Terminai dUTP nick and labelhg (TUNEL) positive cells in the different regions of the brain in normal aging and Alzheimer patients- J. Mol. Neurosci. ; 8: 75-82.

Liu X, Kim CN, Yang J, Jemmerson R, and Wang X. (1996) Induction of apoptotic program in cell fke extracts: requirements for àATP and cytochrome c. Ce14 86: 147- 157.

Loo DT, Copani A, Pike CJ, Whittemore ER, Walencewicz AI, and Cotman CW. (1 993) Apoptosis is induced by beta-arnyloid in cultured central nervous system neurons, Proc. NUL Acad Ski; 90: 7951-7955.

Lorenzo A and Yankner BA. (1 994) Beta-amyloid neurotoxicity requires fibd formation and is inhibited by Congo red. Proc. Nat. Acud Sci.; 91 : 12243- t 2247.

Mann DMA, Iwatsubo T, Caims NJ, et al. (19%) Amyloid ss protein (Ass) deposition in chromosome 14-linked Alzheimer's disease: predominance of Ass42(43). Ann NewoI. ; 40: 1 49- 1 56.

Mann DMA, Mann DM, Iwatsubo T, Cairns NJ, Lantos PL, Nocblin D, Sumi SM, Bird TD, Poorkaj P, Hardy J, Hutton M, Prihar G, Crook R, Rossor MN, Haltia M, et al. (1996) Amyloid beta protein (Abeta) deposition in chromosome l4linked

Alzheimer's disease: predominance of Abeta42(43). Ann N d ; 40(2): 149-1 56.

Mann DMA, Iwaîsubo T, Nochlin D, Sumi SM, Levy-Lahard E, and Bird TD. (1997) Arnyloid (Ass) deposition in chromosome 1-linked Alzheimer's disease: the Volga German families. Ann. Neural. ; 4 1 : 52-57.

Mark RF, Blane EM, and Matison MP. (1996) Amyloid beta-peptide and oxidative cellular injury in Aizheimerys disase. Mol. Neraobiol. ; l2(3): 2 1 1-224.

Mark RI, Hensley K, Butterfield DA, and Mattson MP. (1995) Amyloid beta-peptide impairs ion-motive ATPase activities: evidence for a role in loss of neuronai Ca2+ homeostasis and celi death. J. Netuosci; 15(9): 62394249.

Markesbery WR. (1 997) Oxidative stress hypothesis in Alzheimer's disease. Free Rad Biol. Med; 23(1): 134- 147,

Mariclund SL. (1985) pMogallol autoxidation, in Handbook of Methods for oqgen Radical Resemch, Greenwald, RA (ed). (CRC Press, Boca Raton, Florida), pp. 283-284.

Marx J. (1 998) New gene tied to common form of Alzheimer's. Science; 28 1 : 507-509.

Mano 1, Brenner C, Zamzami N, Jurgensmeier JM, Susin SA, Vieira HL, Revost MC, Xie 2, Matsuyama S, Reed JC, and Kroemer 0. (1 998) Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science; 28 1 : 2027- 203 1.

Masters CL, Simms Gy Weinman N, Mdthaup NA, McDonald Gy and Bayreuther K. (1985 a) Amyloid plaque core pmtein in Alzheimer disare and Down syndrome. Proc. Nat. Acad Sci. ; 82(l2): 42454249.

Masters CL and Bayreuther K. (1988) in Aging ofthe Brain, RD Te- ed, ('aven Press, New York), pp. 1 83-204.

Masters CL, Muithaup NA, Simms G, Pottgiesser J, Martins RN, and Bayreuther K. (1 985 b) Neuronal origin of a cerebral amyloid: neurofibrillary tangles of Alzheimer's disease contain the same pmtein as the amyloid of plaque cores and blood vessels. EMBO J, ; 4: 2757-2763.

Mattson MP. (1994 a) Calcium and neuronal injury in Alzheimer's disease. Contrî'butions of beta-amyloid pecursor protein mismetabolism, fkee radids and metabolic compromise. Ann N. Y. Acad Sci. ; 747: 50-76.

Mattson MP. (1994 b) Secreted f o m of beta-amyloid p r e c m r protein modulate dendrite ouîgmwth and calcium responses to glutamate in cdtured embryonic hippocampal neurons. J. Neurobiol. ; 25(4): 439-450.

Mattson MP. (1997) Cellular actions of beta-amyloid precursor protein and its soluble and fibrillogenic derivatives. PhysioL Rev. ; 77(4): 1 O8 1 - 1 132.

Mattson MP, Barger SW, Cheng B, Lieberburg 1, Smith-Swintosky VL,, and Rydel RE. (1993bl) Beta-amyloid precursor protein metabolites and loss of neuronal Ca2' homeostasis in Alzheimer's disease. Trends N'ewosci. ; 16: 409-414.

Mattson MP, Cheng B, Culwell AR, Esch FS, Lieberburg 1, and Rydel R (1993 a) Evidence for excitoprotective and interneuronal calcium-reguiating d e s for secreted fomis of the m y l o i d precufsor pmtein. Neuron; 10: 243-254.

Mattson MP, Cheng B, Davis D, Bryant K, Lieberburg 1, and Rydel R (1 992) p-amyloid peptides destabilize calcium homeostasis and render humau corticai neurons vulnerable to excitotoxicity. J. Neurosci. ; 12: 376-3 89.

McGeer PI, Rogers I, and McGeer EG. (1994) Neuroimmune mechanisms in Alzheimer disease patients. Alzheimer Dis. Assoc. Disord ; 8: 149- 1 58.

Mecocci P, MacGarvey U, and Beal MF. (1994) Oxidative damage to mitochondrial DNA is increased in Alzheimer's disease. Ana Neurol.; 36: 747-75 1.

Mecocci P, Polidori MC, Ingegni T, Cherubini A, Chionne F, Cecchetti R, and Senin U. (1998) Oxidative damage to DNA in lymphocytes h m AD patients. Newol.; 5 1 : 1014-1017.

Meda L, Cassatella MA, Szendrei GI, et al. (1 995) Activation of micmglia cells by beta- amyloid protein and inteerferon-gamma Nature; 374: 647650.

Meier-Ruge WA and Bertoni-Freddari C. (1 997) Pathogenesis of decrûised glucose turnover and oxidative phospborylaîion in ischemic and trauma-induced dementia of the Alzheimer type. Ann N. Y. Acad Sci ; 826: 229-24 1.

MuUer U, Crisha N, Li ZW, Wolfer DP, Lipp HP, Rulicke T, Brandner S,

Milward EA, Papadopoulos R, Fuller SJ, Mou RD, S m 4 D, Beyreuther K, and Masters CL. (1 992) The amyloid protein precursor of Alzheimer's disease is a mediator of the effects of nerve growth factor on neurite outgrowth. Neuron; 9(1): 129-1 37.

Morris JC. (1995) Relationships of plaques and tangles to Alzheimer's disease phenotype, in Pathobiology ofAlzheimer 's diseuse, Eds. Goate A M and Ashall F.

(Academic press7 New York), pp. 1 9 1 -223.

Mucke L, MasLiah E, Johnson WB, Ruppe MD, Mord M, RockenStein EM, Fross-Petter S, Pietropaolo M, Mdory M, d Abraham CR (1994) Synaptotrophic effects of humau amyloid beta protein precursors in the cortex oftransgenic mice. Brain Res.; 666(2): 15 1 - 167.

Mullan M, Crawford F, Axeiman K, Houlden H, Lilius L, Winblad B, and Landelt L. (1 9%) A pathogenic mutation for probable Alzheimer's disease in the APP gene at the Kîenniuus of &ta-amyioid. Nat. Genet.; l(5): 345-347.

Murphy MP, Hickman LI, Eckman CB, Cnjon SN, Wang R, and Golde TE. (1999) y- secretase, evidence for multiple proteolytic activities and influence of membrane positioniog of substrate on generaîion of amyloid f3 peptides of vaq@ length. J. Biol. Chem; 274(I 7): 1 1914-1 1923.

Murrell J, Farlow M, Ghetti B, and Benson MD. (199 1) A mutation in the amyloid precursor protein associated with hereditary Alzheimer' di-. Science; 254: 97- 99.

Mutisya DM, Bowüng AC, and E3eal MF. (1994) Cortical cytochrome oxidase activity is reduced in Alzheimer's disease. J , Nemchem. ; 63: 2 179-2 1 84.

Mutisya DM, Bowling AC, Waker LC et al. (1993) Impaired energy metabolism in aging and Alzheimer's disease- Neurosci. Absir. ; 1 9: 1474.

Nagi MN, Al-Beeckairi AM, and Al Sawaf HA. (1 995) Spectmphotometric assay for supemxide dismutase based on the nitroblue tetrazoiium reduction by giucose- glucose oxidase. Biochem. Molec. Bio. lnt. ; 36: 633-63 8.

Nalbantoglu J, Tirado-Santiago O, Lahsaini A, Poirier J, Goncalves O, Verge G, Mornoli F, Welner SA, Massictte G, Julien J-P, and Shapiro ML. (1997) Impaired leaming and LTP in mice expressing the carboxy temiinus of the Alzheimer amyloid precursor protein. Nature; 387: 500-505.

Neve RL, Fiuch EA, and Dawes LR. (1988) Expression of the Alzheimer amyloid precursor gene transcnpts in the human brain. Neuron; l(8): 669477.

Oda T, Pasinetti GM, Osterberg HH, Andetson C, Johnson SA, and Finch CE. (1994) Purification and charaçterization of brain clusterin. Biochem. Biophys. Res. Commun.; 204: 11 3 1-1 136-

Olanow CW and Arendash GW. (1994) Metals and fiee radicals in neinodegendon. Curr. Opin Neural. ; 7(6): 548-558.

Olichney JM, Hamen LA, Hofstetter R, Gnindman M, Katzman R, and Thai U. (1995) Cerebrd Uifarction in Alzheimer's disease is associated with severe amyloid angiopathy and hypertension. Arch. NeuroL; 52: 702-708.

Olichney JM, Hansen LA, Galasko D, et al. (1996) The apolipoprotein E e-4 de le is associated with i n d neuritic plaques and cerebral amyloid angiopathy in Alzheimer's disease and the Lewy body variant Naaology; 47: 190-196.

Oltersdorf T, Fritz L, Scheak DB, Lieberburg 1, Johnson-Wood KL, Beattie EC, Ward PJ, Blacker RW, Dovey HF, and Sinha S. (1989) The secreted fomi of the Alzbeimefs arnyloid precursor protein with the Kunitz domain is pmtease nexin-Ii. Nature; 34 1 : 144- 147-

Parker WD, OIey CA, and Padrs K. (19891al) Deficient NADH:coenyme Q oxidoreductase in Leber's hereditary optic neuropathy. N. Eng J Med ; 320: 13 3 1 - 1333.

Parker WD Jr. (1 991) Cytocbme oxidase deficiency in Alzheimer's disease. Ann N. Y- Acad Ski.; 640: 59-64.

Parker WD and Parks IK. (1995) Cytochrome c oxidase in Alzheimer's disease btain: purification and characterization. N m l . ; 45: 482-486.

Parker WD, Boyson SJ, and Padrs K. (1989 b) Abnorrnalities of the electron transport c h d in idiopathic Parkinson's disease. Ana Neurof.; 26: 7 19-723.

Parker WD, Boyson SJ, Luder AS, and Parks K. (1990 b) Evidence for a defect in NADHG;ubiquinone oxidoreductase (cornplex 1) in Huntington's disease. NwoII; 40: 1231-1234.

Parker WD, Filley CM, and P h JK. (1990) Cytochrome oxidase deficiency in Alzheimer's diseasp. Neurology; 40: 1302- 1303.

Parker WD, Mabr NJ, Filley CM, Parks JK, Hughes D, Young D& and Cuiium CM. (1 994 a) Reduced platelet cytochrome c oxidase deficiency in Alzheimer's disease. Neurology; 44: 1086-1090.

Parker WD, Parks JK, Filley CM, and Kleinschmidt-DeMastem BK. (1994 b) Electmn transport chain defecîs in Aizheimer's disease. Neurology; 44: 1090-1 0%.

Partridge W, Munroe SM, Parks K, et al. (1994) Spin trapping of azidyl and hydroxyi radicals in &de-inhibited rat brain submitochoadrial particles. Arck Biochem Bi0phys.i 310: 210-217.

Peilegrini-Giampietro D, Cherici G, Alesiani M, Carla V, and Moroni F. (1988) Excitatory amino acid release h m rat hippocampal slices as a consequerice of fke-radical formation- A Neiaochem.; 5 1 (6): 1 960-1 963.

Perez RG, Squazu, SL, and Koo EH. (1996) Enhanced release of amyloid beta-protein h m codon 670/671 "Swedish" mutant beta-amyloid precucsor protein occm in both secretory and endocytic pathways. J. Bi'ol. Ckm; 271(15): 91ûû-9107.

Petit PX, Susin SA, Zamzami N, Mignote B, and Kroemer G. (1996) Mitochondria and programmeci ce11 death: back to the fiiture. FEBS Lett.; 39q1): 7-1 3.

Pike CJ, Burdick D, Walencewicz AJ, Glaôe CG, and Co- CW. (1993) Neurodegeneration induced by p-amyloid peptides in vitro: The d e of peptide assembly state. J. N ~ o s c i . ; 1 3 : 1676- 1687.

Pike CJ, Walencewicz Al, Glabe CG, and Cotman CW. (1991) in vitro aging of betagmyloid protein causes peptide aggregation and nemtoxicity. Brain Res.; 563(1-2): 3 13-3 14.

Ponte P, Gonzalez-DeWbitt P, Schilling J, Miller J, Hsu D, Greenberg B, Davis K, Wdace W, Lieberburg 1, and Fufler F, (1 988) A new A4 amyloid mRNA contains a domain homologous to serine proteinase inhibitors. Nature; 33 1 : 525-527.

Price DL, Sisodia SS, and Borchelt D R (1998) Genetic neurodegenerative diseases: The human ilhess and transgenic mdels. Science; 282: 1079-1083.

Pnce DL and Sisodia SS. (1998) Mutant genes in familial Alzheimer's disease and transgenic models. Ann, . Rev. Neurosci. ; 2 1 : 479-505.

Pnck MJJ, Gabreels FJM, Trijbels JMF, et al. (1983) Progressive poliodystiophy (Alper's disease) with a defect in cytochmme a% in muscle; a report of two unrelated patients. C h Nevol. Newowg.; 85: 57-70.

Qiu WQ, Ferreira A, Miller C, Koo EH, and SeUcoe DJ. (1995) Ceil-surface beta-amyloid precinsor protein stimulates neurite outgrowth of hippocampal neurons in an isoform-dependent manner. J. Neurosci. 1 S(3 Pt 2): 21 57-2 167.

Reichmann H, Florke S, Hebenstrein G, Schrubar H, and Riederer P. (1993) Analysis of energy metabolkm and mitochondrial gemme in pst-mortem brain fkom patients with Alzheimer's disease. J Neurol ; 240: 3 77-380.

Retz KC and Coyle JT. (1982) Effects of kainic acid on high-energy metabotites in the mouse striatum. J. Neurochem; 38(1): 196.203.

Riviere S, Birlouez-Aragon 1, and Vellas B. (1998) Plasma protein glycation in Alzheimer's disease- Glycoconj- J. ; lS(10): 1039-1042.

Rimito R, Simpson AW, Brini M, aml Pozzan T. (1992) Rapid changes of mitochonârial Ca2+ revealedby specijïcally targeted recombinant aeqw~. N m e ; 358: 325-327.

Roberts GW, Gentleman SM, Lynch A, and Graham DI. (1 99 1) beta A4 amyloid protein deposition in brain d e r head trauma_ Lancet; 338: 142% 1423.

Roch JM, Masliah E, Roch-Levecq AC, Suodsmo MP, Otem DA, Veinbergs 1, and Saitoh T. (1994) Increase of synaptic density and memory retention by a peptide representing the trophic domain of the amyloid betaIA4 protein precufsor. &oc. Nat- A c d Sei,; gl(I6): 7450-7454.

Rogaev EI, Sherrington R, Rogaeva EA, Levesque G, Ikeda M, et al. (1995) Familial Alzheimer's disease in kindreds with missense mutations ina gene on chromosome 1 related to Alzheimer's àisease type 3 gene. Naîwe; 376: 775-778.

Roher A, Wolfe D, Palutke M, and KuKurga D. (1986) M c a t i o n , ultrastructure, and chernical anaiysis of Alzheimer disease amyloid plaque core protein. Proc. Nat. Acad Sei.; 83(8): 2662-2666.

Roth M, Tomlinson BE, and BIessed G. (1 966) Correlation between scores for dementia aad counts of 'senile plaques' in eerebral grey maiter of elderly subjects. Nec; 209: 109-1 10.

Saido TC, Iwatsubo T, Mann DM., Shimada H, Ihara Y, and Kawashima S. (1995) Dominant and differential deposition of distinct beta-amyloid peptide species, A beta N3@E), in senile plaques. N i n ; 14(2): 457-466.

Salbaum SM, Weidemann A, Lemaire HG, Masters CL, and Beyreuther K. (1988) The promoter of Alzheimer's disease amyloid A4 precursor gene. EMBOJ. ; 7(9): 2807- 2813.

Sarnbamurti K, Shioi J, Anderson JP, Papolla Miî, and Robakis NK (1992) Evidence for intracellular cleavage of the Alzheimer's amyioid precursor in PC12 cells. J. Netuosci. Res. ; 33 (2): 3 19-329.

Sambrook J, Fritsch El?, and Maniatis T, (1989) in Molecllm Cloning: A Laborutory Mamal, 2d Edn. Eds: Ford N, Nolan C, and Ferguson M. (Cold Springs Harbor Labotatory Press, Mass).

Scheuner D, protein vivo by

Eckman C , Jeosen M, Song X, Citron M, et al. (1996) Secreted amyloid similar to that in the senile plaques of Alzheimer's disease is i n d in the presenih 1 and 2 and APP mutations linked to familial Alzheimer's

disease. Na- Med ; 2: 864-870.

Schweers O, Mandelkow EM, Biemat J, and Mandelkow E. (1995) Oxidation of cysteine-322 in the repeat domain of microtubule-itssociated protein z controls the in vitro assembly of paired helical filaments. Proc. Nat- Acad Ski.; 92: 8463- 8467.

Selkoe DJ. (1994 a) Alzheimer's disease: a central d e for amyloid. J NewopathoL Exp. NwolI; 53(5): 438-447.

Seikoe DJ- (1994 b) Normal and a b n o d biology ofthe beta-amyioid precursor protein. Annu Rev, Neurosci. ; 17: 489-5 1 7.

Selkoe DJ. (1994 c) Amyloid beta-protein precursor: new clues to the genesis of Alzheimer's disease. C m - Opin Nwobiol. ; 4@): 708-7 16.

Selkoe DJ. (1994 d) CeU biology of the amyloid beta-protein precursor and the mechanism of Aizheimer's disease. Ann Rev. Cell BioL ; 10: 373-403.

Seubert P, Oltersdorf T, Lee MG, Barbour lt, Blomquist C, Davis DL, Bryant K, Fritz LC, Galasko D, Thal W, Lieberburg 1, and Schenk D. (1993) Secretion of be&-amyloid precursor protein cleaved at the amino terminus of îhe beta-amyloid peptide. N w e ; 361: 260-262.

Shapira AHV, Cooper IM, Dexter D, Clark JB, Jenner P, and Marsden CD. (1 990) Mitochondrial complex 1 deficiency in Parkinson's disease. J. Newochem. ; 54: 823-827.

Sheehan JP, Swerdlow RH, Miller S W, Davis RE, Parks JK, Parker WD, and Tuttle JB. (1 997) Calcium homeostasis and reactive oxygen species production in cells transfomied by mitochondria fiom individuals with sporadic Alzheimer's disease. J. Neurosci- ; 1 7(l2): 46 12-4622.

Sherrington R, Rogaev EI, Liang Y, Rogaeva EA, Levesque G, et al. (1995) Clonhg of a gene bearing missense mutations in early-onset familial Alzheimer's disease. Nature; 375: 754-760.

Shigenaga MK, Hagen TM, and Ames BM. (1994) 0xidative damage and mitochondilal decay in aging. Proc. Nat. A c a d SEi. ; 9 1 (23): 1 077 1 - 1 0778.

Shimohama S, Tanino H, and Fujimoto S. (1 999) Changes in caspase expression in Alzheimer's disease: cornparison wiih development and aging. Biochem Biophys. Res- Commun; l6;256(2): 38 1-384.

Shofier JM, Watts RL, Juncos JL, Tomni A, and Wallace DC. (1991) Mitochondrial oxidative phosphorylation defects in Parkinson's disease. A m NewoI.; 30: 332- 339.

Shofier JM, Lott MT, L e m AMS, Seibel P, Balhger S W, and Wallace DC. (1 990) Myoclonic epilepsy and ragged-red fiber disease (MERRF) is associated with a mitochondriai DNA ~ R N A ~ mutation. Ce& 6 1 : 93 1-937.

Shoji M, Golde TE, Ghiso J, Cheung TT, Estus S, S M e r LM, Cai XD, McKay DM, Tmtner R, Frangione B, and Younkîn SG. (1992) Production of the Alzheimer amyloid beta protein by normal proteolytic pmcessing. Science; 258: 126129.

Simmons MA and Schneider CR (1993) Amyloid beta peptides act directiy on single neurons. Neurosci. Lett. ; 1 Sû(2): 1 3 3 - 1 36.

Simonian NA and Hyman BT. (1994) Functional alterations in Alzheimer's disease: selective loss of mitochondriat-encodeci cytochrome oxidase mRNA in the hippocampal formation. NewopathoL Exp. Neurol.; 53(5): 508- 1 2.

Simonian NA and Coyle JT. (1996) Oxidative stress in nedegenerative diseases. Ana Rev. Phannacol. Toxicol- ; 36: 83- 106.

Sisodia SS. (1992) Beta-amyloid precursor pmtein cleavage by a membrane-bound protease. Proc. Nat. Acad Sci. ; W(l3): 6075-6079.

Sisodia SS, Koo EH, Beyreuther K, Unterbeck A, and Price DL. (1990) Evidence that beta-amyloid pmtein in Aizheimer's disease is not derived by nomial p m i n g . Science; 248: 492-495.

Smale G, Nichols NR, Brady DR Finch CE, and Horton WE. (1 995) Evidence for apoptotic ce11 death in Alzheimer's disease. Erp. Nwol.; 133: 225-230.

SmaU DH, Nurcombe V, Moir R, Michaeison S, Monard D, Beyreuther K, and Mastea CL. (1 992) Association and release ofthe amyloid protein precursor of Alzfieimef s disease h m chick brain extracellular matrix. J. Neurosd ; 12(11): 4 143-4 1 50.

Small DH, Nurcombe V, Reed Gy Clarris H, Moi R, Beyreuther K, anci Masters CL. (1994) A heparin-binding domain in the amyloid protein precursor of Alzheimer's disease is involved in the regdation of neurite outgrowth. J. Nezuosci; 14(4): 21 17-2127.

Smith CD, Carney JM, Starke-Reed PE, Oliver CN, Stadtman ER, Floyd RA, and Marksbery WR. (199 1) Excess brain protein oxidation and enzyme dyshction in normal aghg and in Alzheimer disease. Proc Nat. Acad M; 88: 10540-10543.

Smith RP, Higuchi DA, and Bmze GJ. (1990) Platelet coagulation factor Xia-inhi'bitor, a form of Alzheimer amyloid precursor protein, Science; 248: 1 126-1 128.

Smith MA, Taneda S, Richey PL, Miyata S, Yan SD, Stem D, S a p LM, Monnier VM, and Peny G. (1994) Advanced Maillard reaction end produc@ are associated with Alzheimer disease pathology . PTOC. Nat. Acad Sci- ; 9 l(12): 57 10-57 14.

Snyder SW, Ladror US, Wade WS, Wang GT, Barrett LW, Matayoshi ED, Krafft GA, and Holtzmm TF. (1 994) Arnyloid-beta aggregation: selective inhibition of aggregation in mLaures of amyloid with different chah lengths. Biophys. J . ; 67(3): 1216-1228.

Su JH, Deng G, and Cotman CW- (1997) Bax protein expression is increased in Alzheimer's braui: correlations with DNA damage, Bcl-2 expression, and brain pathology. J Neuropathol. Ekp. N d . ; 56:86-93.

Su JH, Cummings BJ, and Cotman CW. (1994) Immunohistochemical evidence for apoptosis in Alzheimer's disease. Neworeporl; S(18): 252992533.

Susin SA, Lorenzo HIC, Zamzami N, Mano 1, Snow BE, Brothers GM, Manpion J y

Jacotot E, Costantini P, Loeffler M, Larochette N, Goodlett DR, Aebersold R, Siderovski DP, Penninger JM, and Kroemer G. (1999) Moleculsr characterization of mitochoadrial apoptosis-hducing factor. Nature; 397: 441-446.

Susin SA, Lorenzo HIC, Zamzami N, Marzo 1, Brenner C, Larochette N, Prevost MC, Alzari PM, and Kroemer G. (1999) Mitochondrial release of caspase-2 and -9 during the apoptotic process. J. Exp. Med ; 189(2): 38 1-394.

Swerdiow R, Parks J, Cassarino Dy Maguire D, Meguire R, Bennett Jr. J, Davis R, and Parker Jr. W. (1997) Cybrids in Alzheimer's Disease: A cellular mode1 of the disease? Neurology; 49: 91 8-925.

Swerdiow R, Marcus DL, Frey W, aad Friedman ML. (1994) Comparative brain glucose and ketone body rnetabolism in Alzheimer's disease. A n J Med Ski-; 308: 141- 144.

TanP RE, Guseiia JF, Watkins PC, Bruns GA, St George-Hyslop P. Van Keuren ML, Paîîerson D, Pagan S, Kurnit DM, and Neve RL. (1 987) Amyloid beta pmtein gem: cDNA, mRNA distributio~, a d genetic linkage near the Alzheimer locus. Skience; 235: 880-884.

Tanzi RE, McClatchey Ai, Lamperti ED, Villa-Koman>ff L, Gusella JF, and Neve RL. (1 988) Pmtease inhibitor domain encodeci by an amyloid protein precuisor mRNA associated with AIzheimer's disease- Nature; 33 1 : 528-30.

Tatton WG and Chaimers-Redman RM. (1998) Mitochondria in neutodegeaerative apoptosis: an opportunity for therapy? A m Neurol. ; 44(3 suppl 1): S 134-S 141.

Terry RD. (1 997) The pathology of Alztieùner's disease: numbers count Cletter]. Amr NWO 2. ; 4 1 : 7.

Terry RD, Masliah E, Salmon DP, Butters N, DeTeresa R, Hill R, Hansen LA, and Katzman R (1991) Physical basis of cognitive alterations in Aizheirner's disease: synapse loss is the major correlate of cognitive impairment A m Newol. ; 30: 572- 580.

niinakaran 0, Teplow DB, Siman R Greenberg B, and Sisodia SS. (1 9%) Metabolism of the "Swedish" amyloid precursor protein variant in neuio2a (N2a) cells. Evidence that cleavage at the "beta-~ecretase~~ site occucs in the golgi apparatus. L BioI- Chern-; 271 : 9390-9397.

Thome J, Komhuber J, Munch G, Schïnzel R, Taneli Y, Zielke B, Rosler M, and Riederer P. (1996) New hypothesis on etiopathogenesis of Alzheimer syndrome. Advanced glycation end products. Nerveruuz! [Abstract]; 67(11): 924-929.

Tienari PJ, Ida N, Ikonen E, Simons M, Weidemaon A, Multhaup G, Masters CL, Doai CG, and Beyreuther K. Tiemari P, et al. (1997) Inaa~ellular and secreted Alzheimer Parnyloid species are generated by distinct mechaniisms in culnved hippocampal neurons. Proc. Nat. Acad Ski. ; 94: 4 1 25-4 1 30.

Trojanowski J and Lee V. (1 994) Paired helical filament tau in Alzheimer's disease. The kinase wnnection. Am. J. Patlr ; 144(3): 449-453.

Turner RS, Suzuki N, Chyung AS, Younkin SG, and Lee VM. (19%) Amyloid and p4, are generated inaa~eilularly in cuitured human neurotls and theü secretion increases wiîh maturation. J I Biol. Chem.; 271 : 8966-8970.

Urmoneit B, Prilailis 1, Wihl G, D'Urso D, Frank R, Heeren J, Beisiegel U, and Rior R (1997) Cerebrovascular smooth muscle cells intemaüze Alzheimer amyloid beta protein via a protein pathway: implications for axebrai amyloid

Vander Heiden MG, Chandel NS, Williamson EK, Schumacker PT, and Thompsoa CB. (1997) B&XL regulates the membrane potential and volume homeostasis of mitochondria. Ce& 9 1 : 627-637.

Van Nostrand WE, Wagner SL, Suzuki M, Choi BH, Farrow JS, Geddes JW, Cotman CW, and Curmingham DD. (1989) Protease nexin-II, a potent antichymoûypsin, shows identity to amyloid beta-protein precursor. Nature; 341 : 546-549.

Vinters HV, Wang ZZ, and Secor DL. (1996) Brain parenchymal and microvascular amyïoid in Aizheimer's disease. Brain Pathol-; 6: 179-195.

Vitek Ml?, Bhattacharya K, Gkndening J M , Stopa E, Vlassara H, Buda R, Mmogue K, and Cerami A. (1994) Advanced glycation end products contribute to amyloidosis in Alzheimer disease. Proc. N& Acad Ski.; 91 (1 1): 4766-4770.

Volicer L and Crino PB. (1990) Involvement of fk radicais in dementia of the Alzheimer type: a hypothesis. Newobiol. Agin8; 1 1 : 567-57f.

Wdace DC (1997), in The MoIecuIat and Genetic Basis of Newologrcal Disease, Rosenberg RN, PNsiner SB, DiMaun, S, Barchi RL, Eds. (Butterworth-Heinemann, Boston), pp. 237-269.

Waiiace DC. (1999) Mitochondrial diseases in man and mouse. Science; 283: 1482- 1488.

Wallace DC, Singh G, Lott MT, et al. (1988) Mitochondrial DNA mutation associated with Leber's hereditary optic neuropathy. N. Eng. J Med ; 242: 1427-1430.

Watt AJ, Pike CJ, Walencewicz-Wassermatl AJ, and Cotman CW. (1994) Ultrastructural analysis ofbeta-amyloid-induced apoptosis in cultured hippocampai neurons. Brain Res.; 661(1-2): 147-156.

Weidemann A, Konig O, Bunke D, Fischer P, Salbaurn JM, Masters CL, and Beyreutber K. (1 989) Identification, biogenesis, and localization of p r e c u ~ r s of Alzheimer's disease A4 amyloid protein. Celk 57(1): 1 15-126.

Weidemann A, Paiiga K, Durrwang U, Reinhard FBM, Scbukert O, Evin G, and Masters C. (1 999) Proteolytic pmcessing of the Alzheimer's disease amyloid precursor protein within its cytoplasmic domain by caspase-like proteases. J. Biol. C h ; 274(9): 5823-5829-

Whittemore ER, Loo DT, and Cotman CW. (1994) Exposure to hydmgen peroxide induces ceii death via apoptosis in cultureci rat cortical neurom. Netao Repor-c S(12): 1485-1488.

Wid-Bode C, Yamazaki T, Capell A, Leimer U, Steiner H, Ihara Y, and Haas C. (1997) Inûaceliular generation and accumulation of AB tenninating at amino acid 42, J. Biol. Chem ; 272: 16085-1 6088.

Williams JH, E h g t o n ML, Lynch MA, and Bliss TV. (1 989) Arachidonic acid induces a long-term activity-dependent enbancement of synaptic transmission in the hippocampus. Nature; 34 1 : 739-742.

Wisniewski KM, Robe A, Zigman W, and Silverman W. (1989) Neurojmthological diagnosis of Alzheimer disease. J. Netuopth. Eip- Ne2aoII; 48(6): MM609.

Wisniewski KJZ, Wisniewski M, and Wen GI. (1985) Occurrence of neuropîhological changes and dementia in Alzheimer's disease in Down's syndrome. Ann Neîuol.; 17: 278-282.

Wong-Riley M, Antuono P, Ho KC, Egan R, Hevner R, Liebl W, Huang 2, Rachel R, and Jones 1. (1 997) Cytochrome oxidase in Alzheimer's disease: biochemicai, histochemical, and immunohistochemical analyses of the visual and other systems. Vision Res. ; 37(24): 3593-3608.

Xia D and Samols D. ( 1 997) Enhancd production and O tigomerization of the 42-residue amyloid P-protein by chinese hamster ovary cellsstabily expressing mutant presinilins. J: BioL Chem.; 272: 7977-7982.

Xu H, Sweeney D, Wang R, Thioakaraa G, Lo AC, Sisodia SS, Greengard P, and Oandy S. (1997) Generation of Alzheimer Pamyloid protein in the tram-Golgi network in the apparent absence of vesicle formation. Prix Nat- Acod Ski.; 94: 374803152.

Yamada T, Sasaki H, Dohura K, Goto 1, and SakaLi Y. (1989) Structure and expression of the aitematively-spliced fonns of mRNA for the mouse homolog of AIzheimer's disease amyloid beta protein precursor- Biochem Biophys. Res. Commun ; 1 S8(3): 906-12.

Yamatsuji T, Matsui T, Okamoto T, Komatnizaki K, Takeda S. Fukumoto H, Iwatsubo T, Suzuki N, Asamiodaka A, Ireland S, Kinane TB, Giambarella U, and Nishimoto 1. (1996 b) G protein-mediated neuronal DNA fkigmentation induced by familial Alzheimer's disease-associated mutants of APP. Science; 272: 13494352.

Yamatsuji T, Okemoto T, Takeda S, Murayama Y, Tanaka N, and Nishimoto 1. (1996 a) Expression of V642 APP mutant causes cellular apoptosis as Alzheimer

trait-linked phenotype. EMBO 1 ; 15(3): 498-509.

Yan SD, Chen X, Fu J, Chen M, Zhu H, Rohei A, Slattery T, Zhao L, Nagashima M, Morser J, Migheli A, Nawmth P, Stern D, and Schmidt AM. (1996) RAGE and amyloid-beta peptide neurotoxicity in Alzheimer's disease. Nature; 382: 685-69 1 .

Yan SD, Chen X, Schmidt AM, Brett Jy Godman Gy Zou YS, Scott CW, Caputo C, Frappier T, Smith MA, et al. (1994) GLycated tau protein in Alzheimer disease: a mechanism for induction of oxidant stress. Proc. Nat- Acad Sei; 9 1 :7787-7791.

Yan SD, Fu J, Soto C, Chen X, Zhu H, AI-Mohama F, Collison K, Zhu A, Stem E, Saido T, Tohyama M, Ogawa S, Roher A, and Stern D. (1997) An intraceliular protein that binds amyloid B-peptide and mediates neurotoxicity in Alzheimer's disease. Nrzftcre; 389: 689-695.

Yang A, Knauer M, Burdick D, and Glabe C. (1 995) intracellular A B 1-42 aggregates stimulate the stable, insoluble amyloidogenic fhgments of the amyloid precursor protein in transfected ceils. J. Biol. C h ; 270: 1 4786- 1 4792.

Yang X, Khosravi-Far Et, Chang YH, and Baltimore D. (1 997) Daxx, a novel fm-binding protein that activates JNK and apoptosis. Cell; 89: 1067-1 076.

Yankner BA. (19%) Mecbanisms of neuronal degeneration in Alzheimer's disease. Netuon; 1 q5): 92 1 -932.

Yankner BA, Dawes LR, Fisher S, Viila-Komamff L. Oster-Gtsnite ML, and Neve RL. (1989) Neurotoxicity of a m e n t of the amyloid precursor associated with Alzheimer's disease. Science; 245: 417420.

Yankner BA, Duffy LK, and Kirschner DA. (1990) Neucotmphic and neurotoxic effects of amyloid P protein: reversal by tachykinin neuropeptides. Science; 250: 279-282.

Yoshikawa K, Aizawa T, and Hayashi Y. (1 99î) Degeneration in vitro of pst-mitotic neurons overexpressing the Alzheimer amyloid protein precursor. Niature; 359: 64- 67.

Zhang L, Zhao B, Yew DT, Kusiak JW, and Roth GS. (1997) Rocessing of Alzheimer's amyloid precursor protein during H,O,-induced apoptosis in human neuronal celis. Biochem. Biophys. Res. Commun; 235: 845-848.

Zhang Y, Marciliat O, Giuiivi C, Ernster L, and Davis KJ. (1990) The oxidative inactivation of mitochondrial electron transport chah components and ATPase. J. Biol. Chem.; 265: 16330-16336.

Zhao B, Chrest FI, Horton WE, Jr., S i d a SS, and Kusiak JW. (1997) Expression of mutant amyloid precursor proteins induces apoptosis in PC 12 ceus. J. NeUrosci- Res. ; 47(3): 253-263.

Zoratti M and Szabo 1. (1995) The mitochondrial permeability transition. Biochim, Biophys. Acta ; 1 24 l(2): 1 39-1 76.