CA2268018A1 - Peptides capable of inhibiting the endocytosis of the app and corresponding nucleotide sequences - Google Patents

Abstract

The invention concerns novel peptide and nucleotide sequences, and their pharmaceutical use. More particularly, it concerns novel peptides capable of inhibiting at least partially the phenomenon of APP endocytosis by intervening at the level of the interaction of the FE65 protein with the cytoplasmic region of the APP.

Description

PEPTIDES CAPABLE OF INHIBITING APP ENDOCYTOSIS AND
CORRESPONDING NUCLEOTIDE SEQUENCES
The present invention relates to new peptide sequences and nucleotides, and their pharmaceutical use. More specifically) the present new peptides capable of inhibiting at least partially the APP endocytosis phenomenon.
The peptide (3 amyloid) the major constituent of the amyloid plaque, is a peptide of about 40 amino acids of 4kDa) derived from the cleavage of fAPP
(Precursor of Amyloid peptide). It accumulates significantly in the brain, not only in Alzheimer's disease but also in Down's syndrome more commonly called Down's syndrome.
APP is a glycosylated protein from 100 to 140kDa, having several domains including a transmembrane region ~, an extracellular domain and a field cytoplasmic. The region of the molecule more specifically concerned with expression of the peptide (3 amyloid partly overlaps the domain transmembrane and extends on the other hand within the extracellular domain. Three forms majority of APP, resulting from alternative splicing, have been characterized. The gene for the APP is located on chromosome 21 and mutations have been identified there in 3 to 5% of patients with familial forms of Alzheimer's disease.
To date, the physiological role of fAPP has not been fully elucidated;
however, it is believed to be involved in synaptic contact, acts to postman title growth regulator) and is neuroprotective.
Recent data from the lifted erection have shown the important role of fAPP endocytosis for the production of the peptide (3 amyloid, and allowed identify sequence elements within the cytoplasmic region) used as endocytosis signals. This is how cells transfected with a construction of cDNA deleted from domain C: cytosolic terminal of fAPP) allow only producing the soluble form of fAPP and do not produce the peptide (3 amyloid. However, in vi vo, we did not yet know the precise nature of events responsible for the activation of fAPP endocytosis and the transduction signals associated with it.

2 Elucidation of the exact role of these;> endocytosis ignals in cells, their mode of operation and their characteristics therefore constitutes a stake major for understanding and (therapeutic approach to the disease Alzheimer's and more generally neurodegenerative diseases.
The present invention results from the demonstration by the applicant that FE65 protein is not just a transcriptional activator in the brain but also interacts in the cytoplasmic region APP
by intervening in the modulation of APP endocytosis.
The present invention results more particularly from the identification and the characterization of particular regions (so-called effector regions) of the FE65 protein) involved in the transduction of fendocytosis activation signals from fAPP. The highlighting the existence of such regions allows us to consider the preparation of new peptides which can be used pharmaceutically.
Within the meaning of the present invention) the name protein FE65 covers the protein as well as all of its homologous forms. By homologous form we intends to designate any protein equivalent to the protein considered) of origin cellular diverse and in particular from cells of human origin, or others organizations, and having an activity of the same type. Homologous shovels can to be obtained by hybridization experiments. Within the meaning of the invention, it suffices that sequence of this type has a significant percentage identity for lead to a physiological behavior comparable to that of the FE 65 protein such as claims.
A first subject of the invention therefore relates to peptides capable to at least partially interfere with the interaction of the protein FE65) or of one of its homologous forms) with the cytoplasmic region of APP.
This interference of a peptide according to the invention can manifest itself under various aspects. The claimed peptide may slow) inhibit or stimulate at least partially the interaction between the FE65 protein or one of its forms counterparts with the cytoplasmic region of APP. Die such peptides are preferably peptides capable ~ '' ~ ntagonize at least partially this interaction.

WO 98I21327 PCTlFR96101775

3 According to a particular mode of (invention, the peptides are capable of binding at the level of the interaction domain between I ~ a FE65 protein or one of its shapes homologs and the cytoplasmic region of fAPP.
More preferably, the peptides of (invention include all or part of the peptide sequence encoding pbur the protein FE65 presented in SEQ ID
N ~ l) SEQ ID N ~ 2 or one of its derivatives.
Within the meaning of the present invention) the term derivative designates any sequence differing from the considered sequence due to a degeneration of the code genetic) obtained by one or more genetic and / or chemical modifications, so that any sequence hybridizing with these seduences or fragments thereof and retaining the ability to interact at the ~ level of interaction between FE65 protein) or one of its counterparts, and the cytoplasmic region of APP. By modification of genetic and / or chemical, we can hear any mutation, substitution, deletion, addition and / or modification of one or more residues. Ix term dêr ~ ivé
also includes the sequences homologous to the sequence considered, derived other cellular sources and in particular cells of human origin, or others organisms) and having an activity of the same type. Such sequences counterparts can be obtained by hybridization experiments. Hybridizations can be made from nucleic acid libraries, using as probe the sequence native or a fragment thereof, under varying conditions of hybridization (Maniatis et al., Cf general techniques of molecular biology ?.
Such derivatives can be generated for different purposes, such as especially that of increasing their therapeutic efficacy or reducing their effects secondary, or that of giving them new properties pharmacokinetics and / or biological.
As a peptide derived from the FE65 protein and homologous forms, it is may include in particular any peptide capable of interacting with the region cytoplasmic fAPP) but carrying an effector region made non-functional. Such peptides can be obtained by deletion, mutation or disruption of this region effector on FE65 protein and homologous forms. Such modifications may to be carried out for example by mutagenesis. in vitro, by introducing elements additional or synthetic sequences, or by deletions or substitutions original elements. When a derivative as defined above is produced, his activity partial inhibitor of FE65 protein binding and forms counterparts on his

4 fixation site on fAPP can be highlighted. Any known technique of those skilled in the art can obviously be used for this purpose.
It can also be fragmf: nts of the sequences indicated above. Of such fragments can be generated in different ways. In particular, they can be synthesized chemically) based on the sequences given in the present asks, using the peptide synthesizers known to (man of the job. They can also be synthesized genetically) by expression in a host cell of a nucleotide sequence coding for the peptide sought. In this case, the nucleotide sequence can be chemically prepared using a synthesizer oligonucleotides, based on the peptide sequence given in the present request and genetic code. The nucleotide sequence can also be prepared from the sequences given in the present application, by clipping enzymatic, ligation, cloning, etc.) according to techniques known to (man of profession, or by screening DNA libraries with probes developed from these sequences.
Furthermore, the peptides of (invention namely capable of slowing or at least partially inhibit (interaction between the FE65 protein and shapes homologs and the cytoplasmic region of fAPP may also be peptides having a sequence corresponding to the interaction site of the FE65 protein and of homologous forms on the cytoplasmic region of APP.
Other peptides according to the invention are the peptides capable of entering into competition with the peptides defined above for interaction with their target cellular. Such peptides can be synthesized in particular on the basis of the sequence of the peptide considered, and their ability to compete with the peptides defined above can be determined.
Another object of the invention is antibodies or fragments polyclonal or monoclonal antibodies directed against a peptide such as defined above. Such antibodies can be generated by known methods of (man of career. In particular) these antibodies can be prepared by immunization of a animal against a peptide of (invention, blood collection, and isolation of antibody. These antibodies can also be generated by preparation hybridomas according to the techniques known to (man of:! 'art.
More preferably, the antibodies or antibody fragments of (invention have the ability to partially inhibit sweat (interaction of peptides WO 98I21327 PCT / FR96l01775 claimed with the cytoplasmic region of f APP. They can thus be used for modulate APP fendocytosis.
Furthermore, these antibodies can also be used to detect and or assay fAP'P expression or overexpression in samples organic) and

5 this fact, to inform about its activation state.
The invention also provides non-peptide or non-peptide compounds.
exclusively peptides which can be used pharmaceutically. It is indeed possible, at from the active protein motifs described in the present application, from perform molecules that inhibit the protein-dependent signaling pathway FE65 no exclusively peptide and compatible with pharmaceutical use.
The present invention also relates to any nucleotide sequence coding for a peptide according to the invention. It may in particular be a sequence comprising all or part of the sequence presented in SEQ ID N ~ 1, SEQ ID N ~ 2 or one of their derivatives. By derivative sequence) is understood within the meaning of this invention any sequence hybridizing with the sequence presented in SEQ ID N ~ 1 or in SEQ ID
N ~ 2 or with a fragment thereof and coding for a peptide according to the invention as well that the sequences resulting from these by degeneration of the code genetic.
The different nucleotide sequences of the invention can be of origin artificial or not. These may be genomic sequences) of cDNA, RNA, hybrid sequences or synthetic or semi-synthetic sequences. These sequences can be obtained either by screening of DNA libraries (cDNA library, bank of genomic DNA), either by chemical synthesis or by mixed methods including chemical or enzymatic modification of sequences obtained by screening for banks.
Such nucleotide sequences can be used for production peptides of the invention. The present application thus relates to a method of preparation of such a peptide according to which a cell containing a sequence nucleotide according to (invention, under conditions of expression of said sequence and the peptide produced is recovered. In this case, the party coding for said peptide is generally placed under the control of signals allowing its expression in a cell host. The choice of these signals (promoters, terminators) sequence "leader"
secretion, etc.) may vary depending on the cell host used. By elsewhere, nucleotide sequences of (invention can be part of a vector which may be with autonomous or integrative replication. More particularly, vectors with replication

6 standalone can be prepared using replicating sequences autonomous at the chosen host. As regards integrative vectors, these can be prepared by example using sequences homologous to certain regians in the genome of (host, allowing, by homologous recombination, integration of the vector.
The cellular hosts usable for the production of the peptides of the invention recombinantly are both eukaryotic and prokaryotic hosts.
From suitable eukaryotic hosts) include animal cells, yeasts) or mushrooms. In particular, in the case of yeasts, mention may be made of yeasts of the kind Saccharomyces) Kluyveromyces) Pichia, Sohwanniomyces) or Hansenula. Regarding of animal cells, mention may be made of COS cells, CHO) C127, etc. Among the mushrooms, there may be mentioned more particularly ~~ nent Aspergillus ssp. or Trichoderma ssp.
As prokaryotic hosts, we prefer to use the following bacteria (E.coli) Bacillus) or Streptomyces.
The nucleic acid sequences <> according to the invention can also be used at the production of antisense oligonucleotides or genetic antisense usable as pharmaceutical agents. The antisense sequences are oligonucleotides of small size, complementary to the strand coding for a given gene, and therefore capable to hybridize specifically with transcribed mRNA) inhibiting its translation into protein.
The invention has thus for object the antisense sequences capable of inhibiting at least partially the interaction of FE65 proteins on the cytoplasmic region of APP. Of such sequences may consist of all or part of the sequences nucleic defined above. These are generally sequences or fragments of sequences of sequences coding for peptides interacting with the region fAPP cytoplasmic. Such oliganucleotides can be obtained by fragmentation, etc., or by chemical synthesis.
The claimed sequences can be used in therapy genes, for the transfer and expression in vivo of antisense sequences or peptides able to modulate (interaction of FE65 proteins with the region cytoplasmic fAPP. In this regard) the sequences can be incorporated into vectors viral or non-viral, allowing their administration in vi vo (Medicine and Sciences

7 (1991) 705). As viral vectors in accordance with the invention, it is possible to particularly cite vectors of the adenovirus type) retrovirus, adeno-associated virus or virus (herpes. The present application also relates to recombinant viruses defective comprising a heterologous nucleic sequence coding for a polypeptide according to The invention.

The invention also allows the realization of nucleotide probes, synthetic or not) capable of hybridizing with the nucleotide sequences defined above) usable in the context of gene therapy. Such probes can be used in vitro as a diagnostic tool) for the detection of the expression or overexpression of fAPP, or for the detection of anomalies genetic (poor splicing, polymorphism, point mutations, etc.). These probes can also be used for highlighting and (isolating sequences acids homologous nucleic acid encoding peptides as defined above, at from other cellular sources and preferably from cells of human origins.
The probes of the invention generally contain at least 10 bases) and they can for example include up to (all of the above sequences or of their complementary strand. Preferably) these probes are, prior to their use, marked. For this, different techniques known to those skilled in the art job can be used (radioactive labeling) enzymatic) etc).
The invention also relates to any pharmaceutical composition comprising as active principle at least one peptide as defined above.
It also relates to any pharmaceutical composition comprising as active ingredient at least one antibody and / or an antibody fragment such as defined cl-before, as well as any pharmaceutical composition comprising as a principle active at least one nucleotide sequence as defined above.
Furthermore, it also relates to pharmaceutical compositions in which peptides) antibodies and nucleotide sequence defined above are associated with each other or with other active ingredients.
The pharmaceutical compositions according to the invention can be used to modulate the activation of the APl 'proteins and therefore to modulate its endocytosis and the production of peptides (3 amyloidE; more particularly) these compositions pharmaceuticals are intended to modulate (interaction between FE65 proteins and the fAPP cytoplasmic region. Plus I> referentially, these compositions are intended to slow down or at least partially inhibit (interaction of FE65 proteins with the cytoplasmic region of fAI'P. It is more preferably about pharmaceutical compositions for the treatment of diseases neurodegenerative such as Alzheimer's disease and Down's syndrome.
Another subject of the invention is (use of the molecules described above to modulate (activation of fAPP fendocytosis or typing of diseases

8 neurodegenerative. In particular, (invention relates to (use of these molecules to at least partially inhibit activation of APP endocytosis.
Other advantages of the present invention will appear on reading the examples and figures which follow) which should be considered as illustrative and not limiting.
Legends of figs, rres Figure l: Representation of the vector pGAL4DB-CAPP
Figure 2: Representation of plasmid DNAs on gels obtained according to the example 3.
Figure 3: Comparison of FE65 nucleotide fragments of various origins MATERIALS AND TECHNIQUES IMPLEMENTED
1) The yeast strains used5 are:
The YCM strain of the genus S.cerevisiae (M.9Ta, ura3-52, his3-200, ade2-10l, lys2-8D1, trpl -901, leu2-3,1l2, canr, gal4-: i42) ga180-538) URA3:: GALIl10-IacZ, LYS :: GALIl10-HIS3) was used as a screening tool for the fusion of brain by the deu, r hybrid system.
The strain L40 of the genus S.cerevisiae (Mata, his3D200, trpl-901, leu2-3,112, ade2, LYS2 :: (IexAop) 4-HIS3, URA3 :: (IexAop) 8-hacZ, GALA) was used to check them protein-protein interactions when one of the protein partners is merged with LexA protein. The latter is able to recognize the response element LexA
controlling the expression of the LacZ and His3 reporter genes.
They were cultivated on the following culture media :.
Complete YPD medium: -Yeast extract (10g / 1) (Difco) -Bactopeptone (20g / 1) (Difco) -Glucose (20g / 1) (Merck) ç ~
This medium was made solid by the addition of 20 g / l of agar (Difco).
Minimum YNB medium: -Yeast Nitrogen Low ~~ (without amino acids) (6.7g / i) (Difco) - Glucose (20g / 1) (Merck) This medium can be made solid by adding 20 g / l of agar (Difco).
To allow the growth of auxotrophic yeasts on this medium) iI is necessary add amino acids or nitrogen bases, for which they are dependent on 50mg / ml. 100 Ng / ml of ampicillin are added to the middle to to avoid bacterial contamination.
2) The strain of bacterig used is:.
The Escherichia colt strain TG1 of genotype supE, hsd ~ 5, thi, 0 (lac-proAB), F '[tra D36 pro A + B + IacIq lacZ ~, M 15]. It was used as a means of amplification and isolation of recombinant plasmids used.
It was grown on LB medium: -NaCI (5g / 1) (Difco) -Bactotryptone (10g, n) (Difco) -Yeast extract (5g / 1) (Difco) This medium can be made solid by adding 20 g / l of agar (Difco).
Ampicillin was used at 100 ~ g / ml) this antibiotic is used to select bacteria having received the plasmids carrying as a marker the gene for resistance to this antibiotic.
3) The ~ plasmids used are :.
The vector pGBTlO. (Clontech), a 5.4kb shuttle plasmid which has a origin bacterial and yeast replication allowing it to replicate at high number of copies in these two microorganisms. This plasmid contains a multiple site of cloning located downstream of the sequence encoding the binding domain DNA from GALA and upstream of a terminator to form a fusion protein. he contains also the TRP1 gene of S. cerevisiae which makes it possible to complement yeasts of genotype trpl in order to select them if a minimum medium does not contain of tryptophan. This vector carries the ampicillin resistance gene which allows select the bacteria having it on a medium containing ampicillin.
The pGADlO vectew. provided by Clonte ~ ch and which allows expression in the yeast fusion proteins between the GAL4 transactivatew domain and a protein coded 5 by cDNA from a brain bank) inserted at a site EcoRI.
Le vectew pL. ~ X9 (pBTMII6) (Bartel and aI DA Hartley Ed) Oxford University press page 153) of 5kb homologous to pGBTlO which contains a multiple cloning site located downstream of the sequence coding pow the ~ bacterial repressor LexA and upstream of a terminate pow to form a fusion protein.
10 4) olig_onucleotides ~ the synthesis used are :.
CAA GTC GAC CTA GTT CTG CAT CTG CTC (SEQ ID N ~ 3) CAA GAA TTC AAG AAA CAG TAC ACA TCC (SEQ ID N ~ 4) Oligonucleotides which made it possible to obtain the PCR fragment corresponding to the CAPP
with the EcoRI and SaII sites.
The oligonucleotides are synthesized using the Applied System ABI 394-08. They are removed from the synthesis matrix by ammonia and precipitated two times by 10 volumes of n-butanol then taken up in water. The quantification is done by optical density meswe (1D0 corresponds to 30pg / ml).
Preparation of plasmid DNAs.
Large amounts of DNA are prepared using the Preparation Kit fast DNA from Proméga.
Small amounts of DNA are prepared as follows:
bacteria containing the plasmid are cultured for at least 4 hours in 2 ml of middle LB
in a shaker shaker. They are then centrifuged for 2 minutes at 14000 rpm in Ependorf tubes, then the pellet is resuspended in 100p1 of the solution I (50mM glucose, 25mM tarnpon Tris HCl pHB, 10mM EDTA pH8)) lysed with 200p1 of solution II (0.2M NaOH) 1% SDS). The solution of lysis is then neutralized with 150p1 of solution III (3M acetate potassium) 1l.5%
(v / v) glacial acetic acid). After shaking the tubes until a precipitate fluffy, 150pI u a mixture of phenol / Chloroform (50% phenol and 50%
chloroform saturated with water) are added, and the whole is stirred for 30 seconds.
The WO 98I21327 PCTlFR96 / 01775 the aqueous phase containing the DNA is recovered after centrifugation for 2 minutes at 14,000 rpm. The DNA is then precipitated by adding 0.5 volume isopropanol then centrifuged for 5 minutes at 14,000 rprn and air-dried to finally be included in 20p.1 TE RNAse (10m Tris-HCI solution, M and 1mM EDTA with 50 ~ g / mt RNAse).
G) Enzy ~ nati ed amplification of DNA or PC ~ R (Polymerase Chain Reaction).
PCR reactions are carried out in a final volume of 100A in the presence of the DNA template, dNTP (0.2mM), PCR buffer (Tris-HCL pH 8.5 10 mM, LmM MgCl2, 5 mM KCl) 0.01% gelatin)) of 0.5 Elg of each oligonucleotides and 2.5 IU of Taq DNA polymerase Amplifier (Perkin Elmer) with or without formamide (5%). The mixture is covered with 2 g <> one of paraffin oil to limit evaporation of the sample. The device used is the "Crocodile II"
of Appligene.
We used a denaturation temperature of the matrix of 90 ~ C, a hybridization temperature of the ohgonucleotides to the matrix lower than 5 to 10 degrees at the separation temperature of the oligonucleotides and a temperature elongation by the enzyme at 72 ~ C.
7) The ligatures.
All ligation reactions are carried out at + 14 ~ C overnight in a final volume of 10 ~ 1 in the presence of l00 to 200 ng of vector, 0.5 to 2 pg of insert, 40 IU of T4 DNA ligase enzyme (Biolabs) and ur ~ ligation buffer (50 mM Tris-HCl pH
7.8; MgCI ~ 10 mM; 10 mM DTT; ATP 1 rnM). The negative control consists of the vector ligation in the absence of insert.

1 C.
$) The transformation of bacteria by an asmidg is carried out according to the protocol iv The entire ligating volume (10p1) is used to transform the TG 1 bacteria made competent by the method of Chl; ntg et al) (PNAS, 1988 86) 2172-2175).
The TG 1 bacteria are cultured in a liquid LB medium for a few hours in a shaking oven at 37 ~ C) until an OD of 0.6 to 600nm. The medium is then centrifuged at 6000 rpm for 10 min. Bacteria are returned competent by taking up the bacterial pellet with a volume of TSB (LB + medium l00 g / 1 of PEG 4000, 5% of DMSO) 10 mM of lvIgCl2) 10 mM of MgS04) corresponding to 1/10 of the volume of the medium of the initial culture. After incubation at 4 ~ C
for 30 to 60 minutes, 200p.1 of bacteria are brought into contact with the ligation during minutes on ice. After addition of 200p1 of LB) the bacteria are incubated 30 min at 37 ~ C and then spread on an LB + ampicillin medium.

9) The separation and extraction of the DNAs are carried out as follows:
15 The DNA is separated according to their size by electrophoresis. For to do; different gels are used depending on the size of the fragments to separate:
-gels of polyacrylamides precast (Novex) at 6%, 10% and 20% for the separation of small DNA fragments (75 to 500 bp) - 1% agarose gel (Gibco BR.L) in TBE buffer (Tris base 90 mM;
Borate 90mM; 2mM EDTA) for the separation of large DNA fragments (greater than 500bps) - 2% NuSieve agarose gel (FMC Bioproducts) in TBE buffer for the separation of small fragments (less than 500 bps).
Any migration on agarose gel or on polyacrylamide gel is carried out in one TBE buffer and in the presence of a molecular weight marqueta- (1Kb ladder, Gibco BRL). The DNA is mixed with 1/10 of the volume of the deposit blue (200 g / 1 of Ficoll) 0.5 g / 1 of bromophenol blue) 50 mM EDTA) before being deposited on gel. After migration to 100 volts and staining with ethydium bromide (concentration 0.5pg / ml gel), the bands are displayed under the UV lamp.
The extraction of DNA from the band of a ~; el of agarose is carried out by electroelution as follows: The piece of gel containing the DNA fragment is cut out scalpel and put in a dialysis rod closed by two forceps and containing 100 to SOOpI of TBE. The whole is put in an electrophoresis tank where it undergoes a field electric 100 Volts. The DNA, after coming out of the gel, is then purified by two phenol / chloroform extractions followed by two chloroform extractions, then precipitated in the presence of sodium acetate 1), 3M and 2.5 volumes of ethanol absolute.
After centrifugation (5 min at 14,000 rpm) the DNA pellet is dried and then taken up in 20p1 water.

10) Séauença ~ e fluorescent DNA plasmi; dic ~ ues.
The sequencing is done according to the Sanger method using 4 dideoxyribonucleotides having a different fluorescent marker. The incorporation of one of these dideoxyribonucleotides produces a stop in replication by the Taq DNA polymerase to be sequenced. This reaction will give DNA fragments of different sizes) all terminated in 3 'by one of the 4 dideoxyribonuclëotides.
One pg of a plasmid and 4 picomoles of a primer are added to 9.5p1 of a "prenwc"
supplied by Applied Biosystems under the name of Prisme. The final volume must be of 20p1 to carry out a PCR for 25 cycles decomposing into one step denaturation at 96 ~ C for 30 seconds, a hybridization step at 50 ~ C for seconds and an elongation step at 60 ~ C for 4 minutes.
The DNA fragments, obtained after amplification, are purified on a column.
exclusion (Chromaspin-30 from Clontech); and are then dried with Speed Vac.
The whole is taken up in 5N.1 of a mixture formed of 24p1 of EDTA (50mM) and 120p1 deionized formamide. After denaturation at 96 ~ C for 3 minutes, 3 to 5p1 are deposited on an electropharesis gel. The different DNA fragments are separated according to their size and will pass successively in front of a laser reader of the Device 370 DNA sequencer (Applied Biosystems) where the different fluorescents go to be detected.

11) Preparation of plasmids from the brain bank (Clontech ~).
The brain cDNA fusion bank is sold as bacteria. These the latter contain a plasmid pGAD10 containing an insert corresponding to a Human brain cDNA. The cDNAs of this bank are formed thanks to the oligodT technique and the degenerate oligonucleotide technique which allows to have the 5 'parts of the mRNAs which are difficult to obtain by the first technical.
These cDNAs are cloned into the vector pGAI) IO at the EcoRI site.
After verification of the bank's title) 21g1 of bacteria from the bank fusion of brain, put beforehand in 8 m1 of LB, are spread in a non-confluent on a solid medium in order to keep the representativeness of this bank. We so have spread out 16 boxes of 770 cm2 containing LB + ampicillin medium. Colonies appeared are taken for each box with 30m1 of LB + liquid ampicillin. The suspensions obtained are then placed in an Erlenmeyer flask and incubated in a shake at 37 ~ C for 3 hours. DNA is then extracted from these strains by the Maxiprep technique. The DNA concentration ~ will be determined at 260nm.

12) Transformation of the yeast by a ~ lasnnide.
The yeasts previously cultivated in 100m1 of liquid medium are harvested after centrifugation at 3000 rpm for 3 minutes and suspended in 1m1 of water sterile. After centrifugation at 3000 rpm for 3 minutes, the cell pellet is handed suspended in 1 ml of sterile water and then centrifuged again. This operation is repeated again to remove all traces of the culture medium.
Yeasts are then taken up in 1 ml of the transformation solution I (LiAc 0.1M) Tris-HCl pH 7.5 lOmM, 1 mM EDTA). then centrifuged at 3000 rpm for 3 minutes. The cell pellet is taken up again in 1 ml of solution I of transformation. 501 of this suspension of yeasts are put in the presence of 50 g of sperm DNA from salmon and 1 to 5 ~ g of plasmid DNA. 300A of a solution II of transformation (0.1A LiAc, Tris-HCl pH 7.5 lOmM, EDT, ~ 1mM in PEG4ooo 40%) are then added, then the whole is incubated: r at 28 ° C. for 30 minutes. A shock is then applied to the transformation mixture in a bath-married to 40 ~ C for 15 minutes then the whole is centrifuged at 15,000 rpm for 1 min to harvest the cell pellet. This pellet is taken up in 200.I of water then spread over a minimum agar medium which does not contain the amino acids corresponding to markers provided by the transforming plasmid. The yeasts are then updates cultivate for 72 hours at 28 ~ C.
In the particular case of tranfor-rnaticm of yeast by the bank of CDNA
brain, we proceed as follows:

The yeast used contains the plasmid pGAL4DB-CAPP coding for part C-terminus of the APP fused to the DNA binding domain of GAL4. She is cultivated in 250m1 of minimum medium YNB + His + Lys + Ad + Leu at 28 ~ C under stirring up to a density of 10 'celh ~ les / ml. The cells are harvested by 5 centrifugation at 3000rpm for 10 minutes and resumed in 250m1 of water.
After one re-centrifugation of the cell pellet;> t taken up in 100m1 of water and at new centrifugal. The pellet is then taken up in 1 () inl of solution I of transformation and incubated for 1 hour at 28 ~ C with shaking. After centrifugation the cells are at again taken up in 2.5 ml of the transformation solution I, of 100 μl of the bank 10 of brain cDNA and 20 ml of transformation solution II, then incubated for 1 hour at 28 ~ C with stirring. A thermal shock is carried out on this mixed transformation at 42 ~ C for 20 minutt ~ s. Centrifugation (3000 rpm while 5mn) is repeated 3 times in succession) each time taking the pellet with 10 ml of water sterile. The third time the pellet is taken up with 2.5 ml of PBS. So the Toxic PEG
15 for cells was removed. 2.4 m1 of this suspension is used for inoculate 250 m1 of minimum medium containing the amino acids His) Lys, Ad and grown overnight in a shaker at 28 "C. The 100 μl of this suspension remaining are used to verify the efficiency of the transformation; for that dilutions from 102, 10-3 and 10 ~ of this suspension were produced and spread over a minimum medium containing amino acids His) Lys) Ad. After cooking at 28 ~ C for 2 days colonies obtained were counted and the transformation rate is determined in using the formula: "number of colonies x cGlution factor". Night culture is centrifuged (3000 rpm for 5 min) and washed with sterile water twice in a row. The nerve is then taken up in 2.5 ml of water. 2.4 ml, the volume of which is brought to 10m1 in sterile water) are used to seed 10 boxes of 435 cmz containing a medium YNB + Lys + Ad and incubated for 3 days. The remaining 100 pl is used for perform the same operations as when determining the conversion rate) so of determine the rate of amplification of the number of colonies overnight of culture.
DNA (genomic and plasmid) is extracted from yeasts in the way next:.
The value of an average handle of a yeast clone is put in 200p 1 of a solution TELT (Triton X100 2%, SDS 1%, NaCI l ~ DOmM, Tris pH8 lOmM, EDTA 1mM), presence of 3 g of glass beads of 450 μm in diameter and 200 μl of phenol / chloroform. This mixture is vortexed for 15 minutes, then centrifuged for 2 minutes at 14,000 rpm. The supernatant is collected without removing the pancake protein and the DNA contained in this phase is precipitated with 2.5 volumes of ethanol absolute. After centrifugation for 2 minutes at 14,000 rpm) the DNA pellet East dried and taken up in 201 of TEI2NAse. This DNA solution, which corresponds to a mixture of genomic and plasmid DNA, directly used to transform bacteria. Only plasmid DNA is capable of replicating in bacteria and can be analyzed by the miniprel technique>.

13) Activity test for f3 $ alactosidase.
A nitrocellulose sheet is previously deposited on the Petri dish containing individualized yeast clones. Thanks to the adsorption phenomenon will get a true picture of the location of the clones. This sheet is then diving in liquid nitrogen for 30 seconds to burst the yeast and free as well l3galactosidase activity. After thawing) the nitrocellulose sheet is filed) colonies upwards, in another Petri dish containing paper Whatman previously soaked with 1.5m1 of F'BS solution (NaZHP04 60mM) NaHZP04 40mM, KCI lOmM, MgS04 lmM, pH7) and from 10 to 301 of X-Gal (5-bromo-4-chloro-3-indoyl- (3-D-galactoside) at 50 mg / ml of N, N.-dimethylformamide. The box is then placed in an oven at 37 ~ C fern cover ~ ê to avoid drying. The time appearance of the blue color can be very;> variable, from a few minutes several hours. This test must always be done in the presence of a positive control, interaction is known and quickly turns blue.
EXAMPLE 1 Construction of a vector allowing the expression of a protein of fusion between the C-terminal part of the precursor of the amyloid peptide (CAPP) and the DNA binding domain of GAL, 4 Screening a bank using the double hybrid system requires that the region C-APP terminale (CAPP) be fused to the DNA binding domain of the GAL4 transactivating protein. The expression of this fusion protein is carried out thanks to the vector pGBTIO (see materials and methods)) in which we have introduced) li in the same reading frame as the sequence corresponding to the domain of binding to GAL4 DNA (GAL4DB)) the CAPP coding sequence in the sequence presented in SEQ ID N ~ 1.
The 138 bpd DNA fragment corresponding to the last 46 amino acids of APP was obtained by PCR from oligonucleotides (SEQ ID N ~ 3 and N ~ 4) who also allowed us to introduce EcoRI and SaII sites at the extremities of the sequence. The PCR fragment was introduced between the EcoRI and SaII sites of the multisite for cloning the plasmid pGBTlO downstream of the sequence corresponding to GAL4DB
to give the vector pGAL4DB-CAPP (fig. 2).
The construction was verified by DNA sequencing. This verification has us allowed to show that this fragment did not present mutations generated at Classes of the PCR reaction and that it was merged in the same open phase of reading than that of the fragment corresponding to GAL4DB.
EXAMPLE 2: SCREENING OF THE BRAIN FUSION BANK.
Screening of a fusion library makes it possible to identify clones producing of proteins fused to the transacti domain: GAL4 vector, which can interact with our protein of interest. This interaction makes it possible to reconstruct a transactivator which will then be able to induce the expression of the reporter genes His3 and LacZ in the YCM strain.
To carry out this screening, we chose a fusion bank carried out at go of cDNA from the human brain. As this bank was provided to us under form of bacteria, the plasmid DNA of the bank was first of all purified.
2.1) Preparation of the plasmid DNA of a fusion banaue.
Plasmid DNA from the brain AI) Nc library was extracted according to the Clontech protocol ~ (see materials and methods) ~ 11). During this preparation ~ 1 was important to preserve the representativeness of the bank, that is to say, keep the number of independent plasmids which constitute it and which are among 1.2.106 plasmids.

In order to protect us from the loss of plasmids from the bank during this preparation) the batch of plasmid DNA that we have assembled has been obtained at go of a number of isolated bacterial colonies corresponding to slightly more than twice the representativeness of the bank, ie 4,106 colonies.
2.2) Transformation ~~ n ~ ue de cerv <: au and selection by the activity test (~ galactosidase.
When screening it is necessary to preserve the probability that each plasmid independent of the fusion bank is present in at least one yeast in even time as the plasmid GAL4DB-CAPP. To preserve this probability it is important to have a good yeast transformation efficiency. For that we we have chosen a yeast transformation protocol giving a efficiency of 105 cells transformed with pg of DNA. In addition as the cotransformation of the yeast by two different plasmids reduced this efficiency, we preferred use a yeast previously transformed with the plasmid pGAL4DB-CAPP. This strain YCM-CAPP phenotype His-, Lys-, Leu- was transformed by 100pg of DNA
fusion bank plasmid. This amount of DNA allowed us to get after estimation (see materials and methods) 10 'transformed cells, this who corresponds to a number slightly higher than the number of plasmids independent that is the bank. From this result we can think that almost all plasmids from the bank was used to transform the yeasts. The selection of cells transformed, capable of reconstituting a functional GAL4 transactivator, a been made on a YNB + Lys + Ad medium.
From this selection, 1000 clones with a His + phenotype were obtained. Sure these transformants an activity test (3galact ~ osidase was carried out in order to validate by the expression of the other reporter gene) La ~ .Z, this number of clones obtained.
68 of 1000 clones obtained exhibited the double His + phenotype) (3Ga1 + can correspond to a protein-protein interaction.
EXAMPLE 3: ISOLATION OF PLASrVIIDS FROM THE BANK
In order to identify your proteins that can interact with CAPP) we have extracts them fusion bank plasmids contained in the yeasts selected during of 19 ' double hybrid screening. To be able to obtain a large quantity) this isolation requires prior transformation of E.coli with a DNA extract from strains positive yeasts. Like the plasmid of: the bank contained in this extract is a yeast / E. coli shuttle plasmid it can easily replicate in bacterium. To avoid isolating the plasmid GAL4 DB - CAPP which is also present in the yeast we have worked on strains that do not.
Plasmid DNA from bacterial colonies obtained after transformation by yeast DNA extracts were analyzed by digestion with enzymes restriction and separation of AI) N fragments on agarose gel. We have got 3 different restriction profiles on the 5 clones analyzed (7D) 3E) 9A, 3H, 3G) (fig 2}.
These results showed that at least 2 identical plasmids are represented in 2 clones of different yeasts, the 7D clones, as well as the 3H and 3G clones.
The DNA of clone 3E is obtained from strains of the His + and (3GAL +.
EXAMPLE 4: DETERMINATION OF THE SEQUENCE OF INSERTS OF
IDENTIFIED PLASMIDS.
The sequencing was carried out using the oligonucleotide (SEQ ID N ~ 5) complementary to the GAL4TA region. near the insertion site of the bank of brain cDNA, 52 bpd from the EcoRI site.
The comparison of the 3 sequences with the sequences contained in the libraries of GENBank and EMBL (European Molecular Biology Lab} data showed that sequences of cDNAs that were in lca plasmids from strains 9A and 3H
show 87% homology at the nucleic level with the marine gene coding for the FE65 protein, they are represented in SEQ ID N ~ 2) and that the sequence of plasmid from the 7D strain has 60% homology with this same gene (see figure 3).
Analysis of the sequence of the plasmids is: ~ us of strains 9A and 3H indicates that those-these contain two overlapping regions corresponding to the same mRNA.

20 ~

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4O (2) INFORMATION FOR SEQ ID NO :: 2:
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Arg Glu Glu Ser Gln Leu Thr Trp Thr Gly Phe Ala His Gly Glu Gly Phe Glu Asp Gly Glu Phe Trp Lys Asp Glu Pro Ser Asp Glu Ala Pro Met Glu Leu Gly Leu Lys Glu Pro Glu Glu Gly Thr Leu Thr Phe Pro GCT CAG AGC CTC AGC CCA GAG CCG TTG ~~ CC CAA GAG GAG GAG AAG CTT 192 AIa GIn Ser Leu Ser Pro Glu Pro Leu Pro Gln Glu GIu Glu Lys Leu 1 ~ 50 55 60 CCC CCA CGG AAT ACC AAC CCA GGG ATC .AAG TGT TTC GCC GTG CGC TCC 240 Pro Pro Arg Asn Thr Asn Pro Gly Ile Lys Cys Phe Ala Val Arg Ser Leu Gly Trp Val Glu Met Thr Glu Glu Glu Leu Ala Pro Gly Arg Ser Ser Val Ala Val Asn Asn Cys Ile Arg Gln Leu Ser Tyr His Lys Asn Asn Leu His Asp Pro Met Ser Gly Gly Trp Gly Glu Gly Lys Asp Leu Leu Leu Gln Leu Glu Asp Glu Thr Leu Lys Leu Val Glu Pro Gln Ser 3 ~ 130 135 140 Gln Ala Leu Leu His AIa Gln Pro Ile Ile Ser Ile Arg Val Trp Gly Val Gly Arg Asp Ser Gly Arg Glu Arg Asp Phe Ala Tyr Val Ala Arg Asp Lys Leu Thr Gln Met Leu Lys Cys His Val Phe Arg Cys Glu Ala Pro Ala Lys Asn Ile Ala Thr Ser Leu His Glu Ile Cys Ser Lys Ile Met Ala Glu Arg Gly Asn Ala Arg Cys Leu Val Asn Gly Leu Ser Leu 5 ~ 210 215 220 Asp His Ser Lys Leu Val Asp Val Pro Phe Gln Val Glu Phe Pro Ala Pro Lys Asn Glu Leu Val Gln Lys Phe Gln Val Tyr Tyr Leu Gly Asn Val Pro Val Ala Lys Pro Val Gly Val Asp Val Ile Asn Gly Ala Leu Glu Ser Val Leu Ser Ser Ser Ser Arg Glu Gln Trp Thr Pro Ser His 1 ~ Val Ser Val Ala Pro Ala Thr Leu Thr Ile Leu His Gln Gln Thr Glu Ala Val Leu Gly Glu Cys Arg Val Arg Phe Leu Ser Phe Leu Ala Val Gly Arg Asp Val His Thr Phe Ala Phe Ile Met Ala Ala Gly Pro Ala Ser Phe Cys Cys His Met Phe Trp Cys Glu Pro Asn Ala Ala Ser Leu Ser Glu Ala Val Gln Ala Ala Cys Met Leu Arg Tyr Gln Lys Cys Leu Asp Ala Arg Ser Gln Ala Ser Thr Ser Cys Leu Pro Ala Pro Pro Ala Glu Ser Val Ala Arg Gly Val Gly Trp Thr Val Arg Arg Gly Val Gln Ser Leu Trp Gly Ser Leu Lys Pro Lys Arg Val Gly Gly His Thr Pro TGA AGA AGC CCC ACC TTC CCT CCA CCT '1275 * Arg Ser Pro Thr Phe Pro Pro Pro (2) INFORMATION FOR SEQ ID N0: 3:
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CAAGAATTCA AGAAACAGTA CACATCC
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Claims (21)

1. Peptide characterized in that it is capable of interfering at the level of the interaction of the FE65 protein or one of its homologous forms with the region cytoplasm of APP. 2. Peptide according to claim 1 characterized in that the interference is translated by a slowing down, inhibition or stimulation of said interaction. 3. Peptide according to one of claims 1 or 2 characterized in that it is capable of binding at the level of the interaction domain between the protein FE65 or one of its homologous forms and the cytoplasmic region of APP. 4. Peptide according to claim 1, 2 or 3 characterized in that it comprises all or part of SEQ ID No. 1, SEQ ID No. 2 or a derivative of these. 5. Peptide capable of competing with a peptide according to one any one of claims 1 to 4 at the level of the domain of interaction between the FE65 protein or one of its homologous forms and the cytoplasmic region of APP. 6. Non-peptide or non-exclusively peptidic compound capable of modulate the interaction between the FE65 protein and the cytoplasmic region of the APP
obtained by reproduction of the active units of the peptides according to the claims 1 to 5 by non-peptide or non-exclusively peptide structures. 7. Nucleotide sequence coding for a peptide as defined in claim 1 to 5. 8. Nucleotide sequence according to claim 7, characterized in that it is a sequence comprising all or part of SEQ ID No. 1, SEQ
ID N°2 or of a sequence derived therefrom. 9. Sequence according to claim 7 or 8 introduced into a viral vector or not viral. 10. Antisense oligonucleotide of a sequence according to claim 7 or 8. 11. Antibody or antibody fragment characterized in that it is directed versus a peptide according to one of claims 1 to 5. 12. Nucleotide probe capable of hybridizing with a nucleic acid according to one of claims 1 to 5 or with the corresponding mRNA. 13. Pharmaceutical composition comprising as active ingredient at least a peptide according to one of claims 1 to 6 or an antibody or fragment of antibodies according to claim 11 or a nucleotide sequence according to one of claims 7 to 9. 14. Pharmaceutical composition according to claim 13, characterized in that that it comprises a recombinant viral or non-viral vector in which is incorporated a nucleotide sequence according to claim 7 or 8. 15. Pharmaceutical composition according to claim 14, characterized in that that it is a viral vector chosen from retroviruses and adenoviruses. 16. Pharmaceutical composition according to one of claims 13 to 15 intended to modulate the interaction between the FE65 protein and the region cytoplasmic of APP. 17. Pharmaceutical composition according to claim 16 or 17 intended for interfere with the interaction between the FE65 protein and the region cytoplasmic of APP. 18. Pharmaceutical composition according to claim 16 intended for treatment of neurodegenerative diseases. 19. Use of an antibody or antibody fragment according to claim 11 and/or of a nucleotide sequence according to one of claims 7 or 8 for the typing of neurodegenerative diseases. 20. Process for the preparation of a peptide according to any one of claims 1 to 5 characterized in that a cell containing a nucleotide sequence according to claim 7 or 8 under conditions of expression of said sequence and the peptide produced is recovered. 21. Defective recombinant virus comprising a nucleic sequence heterolog coding for a polypeptide according to one of claims 1 to 5.