Aggregation and amyloidogenicity of fragments of the amyloid A4 protein precursor of Alzheimer's disease

NEUROBIOLOGY OF AGING, VOLUME 1 I, 1990 ABSTRACTS OF SECOND INTERNATIONAL CONFERENCE ON ALZHEIMER'S DISEASE BRAIN AMYLOIDOSIS GODFEOID, J.M. MALOTEAUX. Universit~ Catholique de Louvain, Laboratoire de Neurochimie, B-1200 Brussels, BELGIUM. Senile plaques are pathological structures found in brain regions of patients with Alzheimer's disease. The plaque is composed of a central core of amyloid containing the A4 peptide as a major constituent. Recently, several laboratories have identified a predicted sequence of an A4 amyloid peptide precursor (A4 APP). Three transcripts of A4 APP were identified, two of which containing a serine protease inhibitor domain. The proteins made from these mRNAs have a structure of a transmembrane molecule. However, recent studies indicate that the A4 APP can be detected in two forms: a membrane bound form which can be stained with both antibodies against the carboxyl-terminal and the amino-terminal portions of the A4 APP, and a soluble form detected only with the amino-terminal antibodies. A clone isolated from a human brain cDNA library contained the structural sequence for an A4 APP with a serine protease inhibitor domain in which the 208 amino acids the carboxyl-terminal are replaced by 20 non hydrophibic amino acids. The cDNA sequence encoding the extracellular domain of the transmembrane precursor, which is identical to the amino-terminal domain of the secreted protein was cloned in a bacterial expression vector, and the corresponding protein, obtained as a fusion product with B-galactosidase was used as an immunogen for the production of monoclonal antibodies. The complete cDNA sequences encoding the transmembrane or the soluble precursors were cloned in a mammalian expression vector downstream the SV40 promoter. After transfection, COS cells and their culture medium were analysed in immunoblotting using the monoclonal antibodies. Our results indicate that both the secretion and the proteolytic cleavage of a transmembrane precursor lead to the immunodetection of important amounts of soluble proteins in the culture medium.

199 INDUCED

NEURONAL

EXPRESSION

AND

ALTERNATIVE

SPLICING

PATTERN OF THE A M Y L O I D GENE OF A L Z H E I M E R ' S D I S E A S E *Gerhard

K0nigI,Colin.

L.Masters

2 and K o n r a d

Beyreuther

tCenter for Molecular Biology (ZMBH), University of Heidelberg,

l

lm Neuenheimer Feld

282, D-6900 Heidelberg, F.R.Germany. 2Department of Pathology, University of Melbourne, Parkville, Victoria 3052, Aus~alIa. The BA4 amyloid gene of Alzheimer's disease (AD, PAD gene) gives rise to at least four alterna, ive splicing products (APP695, A P P 7 5 1 ,

&PP770, and APP563

mRNA). In order to establish the expression pattern of the amyloidogenic transcripts

in developing

human

brain

and in adult brain,

splicing pattern of the different splice forms in fetal u of individuals without neurological APP695 adult

we

PAD

studied

the

well as in adult brains

disease and of different ages. In fetal brain

mRNA accounts for more than 90% of total APP mRMA. In cortex of

and aged

brains

of

individuals

without

neurological

diseases

APP695

mRNA is downregulated and the relative amounts of APP751 and APP770 mRNA are upregulated

whereby

APP751 mRNA

becomes

as prominent

as

APP695

mRNA. In order to study the expression

of the PAD gene during differentiation

we

used human neuronal cell lines, which can be induced to differentiate in vitro. We analysed the effects

of inducers of differentiation

the splicing pattern of the PAD gene. Differentiation

on the expression

and

of neuronal cells In vitro

is associated with a strong increase of total PAD transcripts. Depending on the inducing

agent,

differentiated

pattern that closely resembles undifferentiated that observed

neuronal

cells

neuronal

cells

exhibit

an

that found in a developing exhibit

a splicing

alternative

splicing

fetal brain, whereas

pattern that is similiar

in adult brain. These lines of evidence

to

provide insight essential

~r

designing

new

301

~proaches

that

will

ultimately

delineate

the alternative

splicing m~hanism of the PAD hnRNA.

2OO A G G R E G A T I O N A N D A M Y L O I D O G ~ I C I T Y OF FRAGI~ERTS OF THE AMYLOID A4 PROTEIN PRECURSOR OF ALZREIMER'S DISEASE "Thomas Dyrks I, Elke M a c k - K ~ h n I, C o l i n M a s t e r s 2 and K o n r a d Beyreuther I

1: ~n~r for ~lecular 8inlay, University Heidelberg. F.R.~rmany 2: Dept. of Pat~l~y, Uni~ity of ~lbour~, Par~ille, Victoria ~52 A m y l o i d BA4 p r o t e i n (B protein) is the earliest molecular pathological hallmark of A l z h e i m e r ' s disease and according to o u r c e n t r a l working h y p o t h e s i s d i r e c t l y r e l a t e d to neuronal d y s f u n c t i o n d u e to its d e p o s i t i o n between neurons. The BA4 sequence is included in the sequences of three known membrane proteins encoded by the PAD g e n e and g e n e r a t e d by alternative splicing (APE695, APP751, and APP770). The N-terminal residues 1-28 of 8A4 c o r r e s p o n d s to the e c t o d o m a i n and the C - t e r m i n a l 15 r e s i d u e s of BA4 to the N - t e r m i n a l part of the 24residue transmembrane d o m a i n of APP. T h e m a j o r s e c r e t o r y APP forms are g e n e r a t e d by i n t r a c e l l u l a r proteolysis of the membrane proteins and include only the APP ectodomain b u t n o t the C - t e r m i n a l 100 r e s i d u e s of the m e m b r a n e and c y t o p l a s m i c domain. These and the minor secretory forms (De Sauvage, F. and Octave, J.N.,(1989)Science,245,651-653) generated by alternative splicing are not amyloidogenic. E x p r e s s i o n of the C-terminal 100 residues (A4-CT, no s i g n a l s e q u e n c e ) of APP p r o t e i n s in a r a b b i t reticulocyte s y s t e m r e s u l t s in i n s o l u b l e , highly a g g r e g a t e d peptides. E x p r e s s i o n of an A4CT construct p r e c e d e d by a s i g n a l s e q u e n c e (SPA4CT) leads to m e m b r a n e inserted A 4 C T proteins with no tendency to a g g r e g a t e . P o s t t r a n s l a t i o n a l r e m o v a l of m e m b r a n e s g e n e r a t e s a g g r e g a t e s . E x p r e s s i o n of both p r e c u r s o r fragments (SPA4CT and A4CT) in HeLa cells results in m o n o m e r i c forms. No a g g r e g a t e s are observed. These results prove that aggregation is an i n h e r e n t property of A4CT. Membrane insertion of A4CT prevents the aggregation. This supports our earlier suggestion on m e m b r a n e d a m a g e being a crucial step in a m y l o i d BA4 formation. E u c a r y o t i c cells are able to prevent a g g r e g a t i o n of these p r e c u r s o r fragments (A4CT and SPA4CT). The underlying mechanism remains obscure.

20] THE ALZHEIMER AMYLOID PRECURSOR IS PRODUCED BY TYPE I ASTROCYTES IN GLIAL CULTURES AND DISPLAYS A FIBRILLARY DISTRIBUTION PATTERN. *N.K. Robakis, L.M. Refolo, D. Casper, M. Blum, F. Berkenbosch, V.L. Friedrich Jr. Mount Sinai School of Medicine, New York, NY 10029 USA. The production of B-amyloid precursor protein (BAPP) in primary g l i a l cell cultures from cerebral cortex of 2-3 day postnatal rats was examined by Northern and Western blotting and by immunocytochemistry. A single BAPP RNA transcript was detected when a probe recognizing all forms of BAPP mRNA was used. No signal was detected with a probe specific for the mRNA which encodes the Kunitz Protease Inhibitor-containing BAPPwas used. Western blot analysis of protein extracts, using antisera specific for either the cytoplasmic or extracellular portions of ~PP revealed the presence of several proteins ranging from 105 to 140 kDa. None of these proteins were recognized by an antiserum specific to the KPI insert of the BAPP. Multicolor immunofluorescence showed BAPP immunoreactivity in type I (GFAP+A2B5-) astrocytes, distributed in a f i b r i l l a r pattern l i k e that of g l i a l f i b r i l l a r y acidic protein. S i n c e BAPP is a transmembrane plasmalemmal protein and the antiserum used for immunostaining is directed against the extracellular portion of BAPP, the detected immunoreactivity could reflect a specialized distribution of BAPP in the plasmalemmal. Alternatively, i t is possible that immune reagents penetrated into the fixed cells, and the f i b r i l l a r distribution of 8APP could be i n t r a c e l l u l a r . Regardless, the data suggest an association of BAPP and cytoskeleton that may bear on the function of this molecule and on the development of pathological amyloid deposits. No BAPP immunoreactivity was detected in oligodendrocytes (GC+) or in A2B5+ progenitor cells. In addition, no cultured cells showed immunostaining with an antiserum specific for the KPI sequence of BAPP.