Presenilin's action on amyloid precursor protein cleavage is mediated through cytoplasmic linker protein 170

Poster Presentation:

S180

)8141 LIGATION INDUCED

OF MICROGLIAL CD40 RESULTS IN SRC KINASEP44l42 MAPK-DEPENDENT TNF-a PRODUCTION.

differentially specific with

Tonwce

Town,

.lun Tan. Daniel

Institute.

Turnpa,

Pans.

Yujuan

Wu. Mrhnel

Mullan,

The Ro>knmp

FL

variations

interaction

(CDK)4,6

shown

on microglia

cells, suggesting

that P-amyloid resulting

an inflammatory

in Alzheimer’s

However,

the intracellular

activation

have yet to be investigated. activation

p44/42

MAPK

disease (AD) signaling

withm

of the upstream

activator

phosphorylation ligation

of microglial

p44/42

MAPK

MAPK

CD40.

Further,

TGF-PI

mediated TNF-u

production

in a statistically

MAPK

activity.

Taken

trigger,

TNF-a

~44142

MAPK,

suggesting

MAPK

with

interactwe

that

cascade may be beneficial

CD40

re-entry

cells with

CD40L

inhibits

CD40-

of microglial

CD40

THE

Several

findingr

&ease

(AD)

P Murphy,

(apoE)

constitutes

the

factor

for AD

Ziani-Cherif: Univ.

Ahdul

Fuuq,

Neh. York, NY:

Mqo

INHIBITION

in A(r) production

previously

an a\

yet

OF

accumulation

of

characterize

activity,

AB

APP

based inhibitors

enzyme(s).

inhibit activity.

production

inhibit

AP42 production

A640

unaltered.

ewymatic

of which

differed

reposition

the major

membrane

junction

correspondingly.

from

pepstatin).

Since

cleavage

affinity

accretion

In

order

studies

on Notch

are

processing.

that are cleaved

baaed wategy

cowrained

activity,

for y-wcretase

production

by pepatatm both by

to inhibitor

treatment

(some

mutants

which

domain from

the cytoplasmic

and insensitive enzyme

sites

has been

for the cleavage

of APP or a reparate

evaluate

prowde

the

further

enzyme,

and also provide

effect

of

support

thex

for

the

and ii) y-secretase a framework

for an

purification.

piJ

Mount

Sinai

Sch

Ho,

Piali

Mukherjee,

of Medicine. New

Shrishailam

Alzheimer’s COX-2 pression amyloid

disease (AD)

as well

wth

of human (AP)

hCOX-2

and excitotoxic

potential in the

COX-2

COX-2

M Pminetti,

Using

activation

cytosolic

and cyclooxygenase-2 pro-inflammatory

and in vitro

in human

pathway

prostaglandin

hnk between

phovpholipase

A2

We report

ex-viva in rat aortae smooth

muscle

E2 and prostaglandin

F2cu

smooth muscle cells, an

MEK1/2,

A&

are

of mitogen

(COX-2).

cerebravascular

prevented by inhibiting

involved

he involved

potentiate?

neuronal

in

role of overex-

the intensity

of p

expression DNA

might

microarmy

have been technique\,

function

p38 MAPK

cholesterol

and it‘s link to Alzheimer’r

are uncertain. Our screening bmding

propertlea

PSI (PSI L) and corresponding tail

or COX-2.

and inflammation.

of Cytoplaamic

Linker

and ionomycin

known

to increase

domains

(PSIL

recognizes (APP).

PS-I

Further, wuctural

single

amino

requirements

acid

modifications

the Notch

differentiated

on the potential

quences, it is imperative pathway.

which

proteolytic

to wt.

pathway.

function

overlaps

can critically processing

Amyloid

signalling

Parallel

and vim-w-so.

It is

may also affect APP processing,

will

define

A

exerting

it\

may

alao be important

in

of PSI

unforqeen

may have an as yet conse-

to intercalate with

studies are investigating


170

production

alterations.

important,

is believed

either with the binding

Protein

or CLIP-

is placed on residue\

to our studies ia that it interacts

that of CLIP-170. interfere

Precursor

to enzymatic

with

are

binding

to the dissoci-

those regions

of the binding

in adults.

that

that CLIP-170

can influence

emphasis

or amenable

of Notch

of their

leading

is developmentally

Notch

and

conditions

It is hypothewed

within

to be addressed. 8.catenin

Of significance

Similar

overexpression

Special

cells. As manipulation

CaZf

thapsigagin

region of either PS-I

ubstitutiona

for binding.

of studies have shown that PSI through

loop of

the amount of mutated-PSI

presumably

of AD.

requires

Accordingly,

and whether their overexpression

for post-translational

terminally

Binding

as a receptor in cargo vesicles carrying

detailed

pathway

of CLIP-l70),

studies aim to define the mmimum

of A&

Impact

In contrast,

reduces production

for their binding

for proteins

large cytoplasmic

I70 in wtro.

as compared

of A&

or the C-terminw

Current

required

(CLIP-170).

for CLIP-

CLIP-170

production

ation of the complex,

when dysfunc-

library

mutants isolated a clone which encodea the C-terminal Protein-170

with

exprrsfion (wt)

treatment of cells in culture, increase\

co-immunoprecipitatea

dwease (AD)

of a brain

to the wdd-type

mutated PSI L haa a higher affinity

region

Based on this evidence we decided

genea whose

toxicity.

an enryme may

mouse model with

that COX-2

neurodegeneration. regulated

(COX)-2, processes,

To further explore the potential

a transgenic

and found

mediated

Guilio

(PSI)

differential

p-catenin

as neuronal

neurodegeneration.

in the brain we generated

to identify involved

mechanisms

Yemul.

York, NY

Several lines of evidence indicate that cyclooxygenase in inflammatory

E

elevated

appears to be a risk

involving

in intact rat aortae and in human cerebrovascular

Pre$enilin’s

the Notch L~J

pathway

and AP appear to stimulate

tional (mutated)

action

Mirjuny,

with

PRESENILIN’S ACTION ON AMYLOID PRECURSOR PROTEIN CLEAVAGE IS MEDIATED THROUGH CYTOPLASMIC LINKER PROTEIN 170.

critical

COX-2 DEPENDENT CELL CYCLE ACTIVITIES IN BRAIN: IMPLICATIONS IN THE CLINICAL PROGRESSION OF ALZHEIMER’S DISEASE DEMENTIA.

Mana

serum cholesterol

~38 MAPK.

(S-LOX)

triggers a similar

effect that can be completely

number

(816(

and is also arsnciated

of. apoE status. We have shown that AP peptides

kinase kinaae (MEKIIZ),

vasoconstriction,

and Alzheimer’s

of the apolipoprotein

that are

and since there is some issue as to

results

elevated

cholesterol

4 allele

these

or leaving

AP production

or farther

to funher

These

Moreover,

5-lipoxygenase

here that cholesterol

between

better

As with pepstatin, cleavage sites

that i) there are more than one y-secretase

is a spatially

increasing

by a y-secretax-like

responsible

underway

an

a series of substrate

of the inhibitor-sensitive

of this protein,

death by promotmg

and an

to

We then evaluated a subset 01

tranamembrane closer

of Notch

function

this is the same enzyme additional

Also.

AD

both from whole cells and m

sensitivites

site either

alter the position

in the normal

hypotheses

cells.

and examined

spectrometry.

neuronal

the epsilon

rirk factor for AD,

independently

protein

is able to act as an

either alightly

APP constructs

differential

cleavage

of

have

data from the in vitro assay has also indicated

and imlnunoprecipltationmasa

cleavage

while

activities,

in these constructs also exhibited

compounds

and others

We have found several compound!,

Preliminary

in cells expressing

or insensitive

entity,

intact

and A@42 cleavages can be diaaociated.

these compounds

whether

peps&tin

AP production

able to aelectively

implicated

group

we have recently synthesized

an in ~,itrn assay of y-secretase

ELISA

in

We

of

research in

for the generation

with greater potency than pepstatm. At low p,M concentrations.

are able to effectively

that the AP40

Our

a major

These data suggest a pathologic

by a decrease in AP

fragments

Chewki

FL

responsible

proteaae inhibitor

C-terminal

E Smith.

FL; Rang Wang, The Rock<,fel/er Jwkson~ill<~,

actwitiea

as defined

the y-secretaae activity,

compounds

xwtive

C/m,

Tmqu

ia a [major avenue of therapeutic

unknown

shown that the a.\partyl of y-secretase

A Findlq,

Clin. Jorksom~ill~~.

d&ease. One of the proteolytic

A@ is y-secretasr. inhibitor

Kirk

Todd E Gold<,, M
A targeted reduction Alzheimer’s

C McLendon,

For instance,

vasoactive and stimulate a pro-inflammatory

of AD.

inducing

Daniel

dementia.

potentiate

have suggested a relationship

pathology.

cells. Cholesterol Michael

(and

progression

into the cell cycle.

serum total cholesterol.

of Src and dnwmtream at downregulating

in the treatment

FOR

may

of CDK4

decreases

and ultimately

TARGETS

COX-2

clinical

kmase

brain the expression

CHOLESTEROL AS WELL AS AB INDUCES VASOCONSTRICTION BY STIMULATING A SIMILAR PRO-INFLAMMATORY PATHWAY

pig

activated

MOLECULAR PRODUCTION

of

Cell cycle genes that showed

is a cell cycle dependent

Inhibition

manor by decreasing p44/42

aimed

inappropriate

to clusters of

release followmg

or both,

the kinase activity

pharmacotherapy

as a function

that m AD,

in increased

PD98059

of microglial

or IL-IO.

in the cell cycle.

We also found that in AD

increases

and related

of Src kinase activity

these data show that ligation

release through increasing

microglial

of microglial

reduces TNF-u

co-treatment

cytokines

ldentlfied

III

neurode-

induced by CD40L.

MEKI/2,

were

Biology

286:2352-2355).

resulting

Blockade

and significantly

together,

Science

and Lck

activity

MAPK.

and the anti-inflammatory

Src/p44/42

Lyn

mediate

fold)

and Cellular

One cluster was found to have pattern7 that correlated

with genes involved

inhibitor.

hypothewe

levels by these

CDilOL-induced

We report that ligation

of p44/42

of p44/42

mediating

30 min port-treatment.

results in decreased microglial

microglia

[Tan et al. (1999),

kinases

the CD40-CD40L

at neurotoxic

by which

events

of the Src-family

activity

can enhance

secretion

mechanism

generation

triggers

peptldes

in TNF-a

genes (>2

variations.

altered expressmn m this cluster include ~18, which

COX-2) WC have recently

regulated

biological

Molecular

of PS-I

to CLIP-170

that activation

as the two pathways

PSI

to

PSL at a whether or APP

of the Notch

share PSI.