EP0941319A1

EP0941319A1 - Peptides capable of inhibiting the endocytosis of the app and corresponding nucleotide sequences

Info

Publication number: EP0941319A1
Application number: EP96938292A
Authority: EP
Inventors: Luc Merken; Alain Fournier
Original assignee: Rhone Poulenc Rorer SA
Current assignee: Aventis Pharma SA
Priority date: 1996-11-08
Filing date: 1996-11-08
Publication date: 1999-09-15
Also published as: WO1998021327A1; JP2001503988A; CA2268018A1

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 NUCLEOTIDIOUE SEQUENCES

The present invention relates to new peptide and nucleotide sequences, and their pharmaceutical use. More particularly, the present invention relates to new peptides capable of at least partially inhibiting the phenomenon of APP endocytosis.

The amyloid β peptide, the major constituent of the amyloid plaque, is a peptide of about 40 amino acids of 4kDa, resulting from the cleavage of APP (Precursor of

Amyloid peptide). It accumulates significantly in the brain, not only in Alzheimer's disease but also in Down syndrome, more commonly known as Down's syndrome.

APP is a glycosylated protein from 100 to 140kDa, having several domains including a transmembrane region, an extracellular domain and a cytoplasmic domain. The region of the molecule more specifically concerned with the expression of the amyloid β peptide partly overlaps the transmembrane domain and also extends within the extracellular domain. Three major forms of APP, resulting from alternative splicing, have been characterized. The APP gene 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 APP has not been fully elucidated; however, it is believed to be involved in synaptic contact, acts as a growth regulator, and is neuroprotective.

Recent data from the literature have shown the important role of APP endocytosis for the production of the amyloid β peptide, and have made it possible to identify elements of sequence, within the cytoplasmic region, used as signal signals. endocytosis. Thus cells transfected with a cDNA construct deleted from the cytosolic terminal C domain of APP, only allow the production of the soluble form of APP and do not produce the amyloid β peptide. However, in vivo, we do not yet know the precise nature of the events responsible for the activation of APP endocytosis or the transduction of the signals associated with it. The elucidation of the exact role of these endocytosis signals in cells, their mode of operation and their characteristics therefore constitutes a major challenge for the understanding and the therapeutic approach of Alzheimer's disease and more generally of neurodegenerative diseases .

The present invention results from the discovery by the applicant that the protein FE65 does not only constitute a transcriptional activator in the brain but that it also interacts at the level of the cytoplasmic region of APP by intervening in the modulation of the APP endocytosis.

The present invention results more particularly from the identification and characterization of particular regions (so-called effector regions) of the FE65 protein, involved in the transduction of signals for the activation of APP endocytosis. The demonstration of the existence of such regions makes it possible to envisage the preparation of new peptides which can be used pharmaceutically.

Within the meaning of the present invention, the name protein FE65 covers the protein itself as well as all of its homologous forms. The term “homologous form” is intended to denote any protein equivalent to the protein under consideration, of diverse cellular origin and in particular derived from cells of human origin, or of other organisms, and having an activity of the same type. Such homologous sequences can be obtained by hybridization experiments. Within the meaning of the invention, it is sufficient for a sequence of this type to have a significant percentage of identity to lead to physiological behavior comparable to that of the protein FE 65 as claimed.

A first object of the invention therefore relates to peptides capable of interfering at least partially at the level of the interaction of the protein FE65, or one of its homologous forms, with the cytoplasmic region of APP.

This interference of a peptide according to the invention can manifest itself in different aspects. The claimed peptide can slow, inhibit or at least partially stimulate the interaction between the protein FE65 or one of its homologous forms with the cytoplasmic region of APP. Such peptides are preferably peptides capable of at least partially antagonizing this interaction. According to a particular embodiment of the invention, the peptides are 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.

More preferably, the peptides of the invention comprise all or part of the peptide sequence coding for the protein FE65 presented in SEQ ID No. 1, SEQ ID No. 2 or one of its derivatives.

Within the meaning of the present invention, the term derivative designates any sequence differing from the sequence considered due to a degeneration of the genetic code, obtained by one or more modifications of a genetic and / or chemical nature, as well as any sequence hybridizing with these sequences or fragments thereof and retaining the ability to interact at the level of the interaction between the protein FE65, or one of its homologs, and the cytoplasmic region of APP. By modification of genetic and / or chemical nature, one can hear any mutation, substitution, deletion, addition and / or modification of one or more residues. The term derivative also includes sequences homologous to the sequence considered, derived from other cellular sources and in particular from cells of human origin, or from other organisms, and having an activity of the same type. Such homologous sequences can be obtained by hybridization experiments. Hybridizations can be carried out from nucleic acid libraries, using the native sequence or a fragment thereof as probe, under variable hybridization conditions (Maniatis et al., Cf. general techniques of molecular biology).

Such derivatives can be generated for different purposes, such as in particular that of increasing their therapeutic efficacy or reducing their side effects, or that of conferring on them new pharmacokinetic and / or biological properties.

As a peptide derived from the FE65 protein and homologous forms, mention may in particular be made of any peptide capable of interacting with the cytoplasmic region of APP, but carrying an effector region made non-functional. Such peptides can be obtained by deletion, mutation or disruption of this effector region on the protein FE65 and homologous forms. Such modifications can be made, for example, by in vitro mutagenesis, by introduction of additional elements or synthetic sequences, or by deletions or substitutions of the original elements. When a derivative as defined above is produced, its activity as a partial inhibitor of the binding of the protein FE65 and of the homologous forms on its APP binding site can be highlighted. Any technique known to those skilled in the art can obviously be used for this purpose.

They can also be fragments of the sequences indicated above. Such fragments can be generated in different ways. In particular, they can be synthesized chemically, on the basis of the sequences given in the present application, using the peptide synthesizers known to those skilled in the art. They can also be synthesized genetically, by expression in a cellular host of a nucleotide sequence coding for the peptide sought. In this case, the nucleotide sequence can be prepared chemically using an oligonucleotide synthesizer, on the basis of the peptide sequence given in the present application and the genetic code. The nucleotide sequence can also be prepared from the sequences given in the present application, by enzymatic cleavages, ligation, cloning, etc., according to techniques known to those skilled in the art, or by screening DNA libraries with elaborate probes from these sequences.

Furthermore, the peptides of the invention, namely capable of slowing down or at least partially inhibiting the interaction between the protein FE65 and homologous forms and the cytoplasmic region of APP can also be peptides having a sequence corresponding to site of interaction of the protein FE65 and homologous forms on the cytoplasmic region of APP.

Other peptides according to the invention are the peptides capable of competing with the peptides defined above for the interaction with their cellular target. 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 subject of the invention resides in antibodies or fragments of polyclonal or monoclonal antibodies directed against a peptide as defined above. Such antibodies can be generated by methods known to those skilled in the art. In particular, these antibodies can be prepared by immunizing an animal against a peptide of the invention, drawing blood, and isolating the antibodies. These antibodies can also be generated by preparing hybridomas according to techniques known to those skilled in the art.

More preferably, the antibodies or antibody fragments of the invention have the ability to at least partially inhibit the interaction of peptides claimed with the cytoplasmic region of the APP. They can thus be used to modulate the endocytosis of APP.

Furthermore, these antibodies can also be used to detect and / or measure the expression or overexpression of APP in biological samples, and therefore, to provide information on its state of activation.

The invention provides ^an equal ment of non-peptidic compounds or non-peptidic pharmaceutically usable exclusively. It is indeed possible, from the active protein motifs described in the present application, to produce molecules which inhibit the signaling pathway dependent on the FE65 protein which are not exclusively peptide and which are 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 No. 1, SEQ ID No. 2 or one of their derivatives. By derivative sequence is meant within the meaning of the present invention any sequence hybridizing with the sequence presented in SEQ ID No. 1 or in SEQ ID No. 2 or with a fragment thereof and coding for a peptide according to the invention, as well as the sequences resulting from these by degeneration of the genetic code. The different nucleotide sequences of the invention can be of artificial origin or not. D can be genomic sequences, cDNA, RNA, hybrid sequences or synthetic or semi-synthetic sequences. These sequences can be obtained either by screening of DNA libraries (cDNA library, genomic DNA library), or by chemical synthesis, or by mixed methods including chemical or enzymatic modification of sequences obtained by screening of libraries.

Such nucleotide sequences can be used for the production of the peptides of the invention. The present application thus relates to a process for the preparation of such a peptide according to which a cell containing a nucleotide sequence according to the invention is cultivated, under conditions of expression of said sequence and the peptide produced is recovered. In this case, the part coding for said peptide is generally placed under the control of signals allowing its expression in a cellular host. The choice of these signals (promoters, terminators, "leader" secretory sequence, etc.) can vary depending on the cell host used. Furthermore, the nucleotide sequences of the invention can be part of a vector which can be autonomously replicating or integrative. More particularly, replication vectors Autonomous can be prepared using autonomous replicating sequences in the chosen host. As regards integrative vectors, these can be prepared for example by using sequences homologous to certain regions of the host genome, allowing, by homologous recombination, the integration of the vector. The cellular hosts which can be used for the production of the peptides of the invention by the recombinant route are both eukaryotic and prokaryotic hosts. Among suitable eukaryotic hosts, there may be mentioned animal cells, yeasts, or fungi. In particular, as regards yeasts, mention may be made of yeasts of the genus Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces, or Hansenula. As regards animal cells, mention may be made of COS, CHO, C127 cells, etc. Among the mushrooms, there may be mentioned more particularly Aspergillus ssp. or Trichoderma ssp. As prokaryotic hosts, it is preferred to use the following bacteria E.coli, Bacillus, or Streptomyces.

The nucleic acid sequences according to the invention can also be used for the production of antisense oligonucleotides or of genetic antisense usable as pharmaceutical agents. The antisense sequences are small Ogonucleotides, complementary to the coding strand of a given gene, and therefore capable of hybridizing specifically with the transcribed mRNA, inhibiting its translation into protein. The subject of the invention is therefore the antisense sequences capable of at least partially inhibiting the interaction of the FE65 proteins on the cytoplasmic region of APP. Such sequences can consist of all or part of the nucleic acid sequences defined above. They are generally sequences or fragments of sequences complementary to sequences coding for peptides interacting with the cytoplasmic region of APP. Such Ogonucleotides can be obtained by fragmentation, etc., or by chemical synthesis.

The claimed sequences can be used in the context of gene therapies, for the transfer and expression in vivo of antisense sequences or of peptides capable of modulating the interaction of the FE65 proteins with the cytoplasmic region of APP. In this regard, the sequences can be incorporated into viral or non-viral vectors, allowing their administration in vivo (Medicine and Sciences 7 (1991) 705). By way of viral vectors in accordance with the invention, mention may very particularly be made of vectors of the adenovirus, retrovirus, adeno-associated virus or herpes virus type. The present application also relates to defective recombinant viruses comprising a heterologous nucleic sequence coding for a polypeptide according to the invention. The invention also allows the production 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 APP, or even for the detection of genetic anomalies (poor splicing, polymorphism, point mutations, etc.). These probes can also be used for the detection and isolation of homologous nucleic acid sequences coding for peptides as defined above, from other cellular sources and preferably from cells of human origin. The probes of the invention generally comprise at least 10 bases, and they can for example comprise up to the entirety of one of the abovementioned 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 can be used (radioactive, enzymatic labeling, 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 as defined above, as well as any pharmaceutical composition comprising as active ingredient at least one nucleotide sequence as defined above before.

Furthermore, it also relates to pharmaceutical compositions in which the 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 APP proteins and therefore to modulate its endocytosis and the production of amyloid β peptides. More particularly, these pharmaceutical compositions are intended to modulate the interaction between the FE65 proteins and the cytoplasmic region of APP. More preferably, these compositions are intended to slow down or at least partially inhibit the interaction of the FE65 proteins with the cytoplasmic region of APP. More preferably, these are pharmaceutical compositions intended for the treatment of neurodegenerative diseases such as, for example, Alzheimer's disease and trisomy 21.

Another subject of the invention is the use of the molecules described above to modulate the activation of APP endocytosis or the typing of diseases neurodegenerative. In particular, the invention relates to the 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 the figures:

Figure 1: Representation of the vector pGAL4DB-CAPP

Figure 2: Representation of plasmid DNAs on gels obtained according to Example 3.

Figure 3: Comparison of FE65 nucleotide fragments of various origins

MATERIALS AND TECHNIQUES IMPLEMENTED

1) The yeast strains used are:

The YCM strain of the genus S.cerevisiae (MATa, ura3-52, his3-200, ade2-101, lys2- 801, trpl-901, leu2-3,112, canr, gal4-542, gal80-538, URA3 :: GALlll0- lacZ,

LYS :: GAL1I10-HIS3) was used as a tool to screen the brain fusion bank by the two hybrid system.

The L40 strain of the genus S.cerevisiae (Mata, his3D200, trpl-901, leu2-3,112, ade2,

LYS2 :: (lexAop) 4-HIS3, URA3: :( lexAop) 8-LacZ, GAL4) was used to verify protein-protein interactions when one of the protein partners is fused to the LexA protein. The latter is capable of recognizing the LexA response element controlling the expression of the reporter genes LacZ and His3.

They were cultivated on the following culture media :.

Complete YPD medium: -Yeast extract (lOg 1) (Difco)

-Bactopeptone (20g / l) (Difco) -Glucose (20g / l) (Merck) This medium was made solid by the addition of 20 g _/ agar (Difco).

MiUeu YNB rninimum: -Yeast Nitrogen Base (without amino acids) (6.7g l) (Difco)

- Glucose (20g / l) (Merck) This medium can be made solid by adding 20g / l of agar (Difco).

To allow the growth of auxotrophic yeasts on this medium, it is necessary to add to it amino acids or nitrogen bases, on which they are dependent at 50 mg / ml. 100 μg / ml of ampicillin are added to the medium in order to avoid bacterial contamination.

2) The strain of bacteria used is:.

The Escherichia coU TGl strain of genotype supE, hsdΔ5, thi, Δ (lac-proAB), F '[tra D36 pro A ⁺ B ⁺ lacI ^q lacZΔM15]. It has been used as a means of ampUfication and isolation of the recombinant plasmids used.

It was cultivated on LB medium: -NaCl (5g / l) (Difco) -Bactotryptone (lOg / l) (Difco)

-Yeast extract (5g l) (Difco) This medium can be made solid by adding 20g / l of agar (Difco).

Ampicillin was used at 100 μg / ml, this antibiotic is used to select the bacteria which have received the plasmids carrying as marker the gene for resistance to this antibiotic.

3) The plasmids used are :.

The vector pGBTIO. (Clontech). A 5.4 kb shuttle plasmid which has an origin of bacterial and yeast replication allowing it to replicate with a high number of copies in these two microorganisms. This plasmid contains a multiple cloning site located downstream of the sequence coding for the DNA binding domain of GAL4 and upstream of a terminator to form a fusion protein. It also contains the TRP1 gene from S. cerevisiae which makes it possible to complement yeasts of the trpl genotype in order to select them on a minimum medium containing no tryptophan. This vector carries the ampiciUine resistance gene which makes it possible to select the bacteria possessing it on a medium containing ampiciUine.

The vector pGADIO. supplied by Clontech and which allows the expression in yeast of fusion proteins between the GAL4 transactivating domain and a protein coded by the cDNA originating from a brain bank, inserted at an EcoRI site.

The vector pLex9 (pBTMllό) (Bartel et al DA Hartley Ed, Oxford University press page 153) of 5kb homologous to pGBTIO which contains a multiple cloning site located downstream of the sequence coding for the bacterial repressor LexA and upstream of a terminator to form a fusion protein.

4) the synthetic oGonucleotides used are :.

CAA GTC GAC CTA GTT CTG CAT CTG CTC (SEQ ID N ° 3) C AA GAA TTC AAG AAA CAG TAC ACA TCC (SEQ ID N ° 4)

OUgonucleotides which made it possible to obtain the PCR fragment corresponding to CAPP with the EcoRI and Sali sites. The Ogonucleotides are synthesized on the Applied System ABI 394-08. They are detached from the synthesis matrix with ammonia and precipitated twice with 10 volumes of n-butanol and then taken up in water. The quantification is carried out by measuring the optical density (1DO corresponds to 30 μg ml).

5) Preparation of the plasmid DNAs.

Large amounts of DNA are prepared using the Proméga DNA Rapid Preparation Kit.

Small amounts of DNA are prepared in the following manner: the bacteria containing the plasmid are cultured for at least 4 hours in 2 ml of miUeu LB in a shaker shaker. They are then centrifuged for 2 minutes at 14,000 rpm in Ependorf tubes, then the pellet is resuspended in 100 μl of solution I (50 mM of glucose, 25 mM of Tris HC1 ρH8 buffer, 10 mM EDTA ρH8), lysed with 200 μl of the solution II (0.2M NaOH, 1% SDS). The lysis solution is then neutralized with 150 μl of solution III (3M of potassium acetate, 11.5% (v / v) of glacial acetic acid). After stirring the tubes until a flaky precipitate is obtained, 150 μl u a mixture of phenol / Chloroform (50% phenol and 50% chloroform saturated in water) are added, and the whole is stirred for 30 seconds. The 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 volumes of isopropanol, then centrifuged for 5 minutes at 14,000 rpm and air dried to finally be taken up in 20 μl of TE RNAse (10 mM Tris-HCl solution and ImM EDTA with 50μg / ml of RNAse).

6) Enzymatic amplification of DNA or PCR (Polymerase Chain Reaction).

The PCR reactions are carried out in a final volume of 100 μl in the presence of the DNA template, dNTP (0.2 mM), PCR buffer (Tris-HCL pH 8.5 10 mM, MgCl ₂ ImM, KCl 5 mM , 0.01% gelatin), 0.5 μg of each of the oGonucleotides and 2.5 IU of AmpU Taq DNA polymerase (Perkin Elmer) with or without formamide (5%). The mixture is covered with 2 drops of paraffin oil to limit the evaporation of the sample. The device used is the "Crocodile II" from Appligene. We used a denaturation temperature of the matrix of 90 ° C, a hybridization temperature of the Ogonucleotides to the matrix 5-10 degrees lower than the separation temperature of the Ogonucleotides and an elongation temperature of 72 ° C.

7) The ligatures.

All the ligation reactions are carried out at -f-14 ° C overnight in a final volume of 10 μl in the presence of 100 to 200 ng of vector, 0.5 to 2 μg of insert, 40 IU of T4 DNA Ugase enzyme (Biolabs) and a ligation buffer (50 mM Tris-HCl pH 7.8; 10 mM MgCl ₂ ; 10 mM DTT; 1 mM ATP). The negative control consists of the ugation of the vector in the absence of an insert. 8) The transformation of the bacteria by a plasmid is carried out according to the following protocol :.

The entire volume of Ugation (10 μl) is used to transform the TGl bacteria made competent by the method of Chung et al, (PNAS. 1988 86, 2172-2175). The TGl bacteria are cultured in a liquid LU medium for a few hours in a shaking oven at 37 ° C., until an OD of 0.6 to 600 nm is obtained. The medium is then centrifuged at 6000 rpm for 10 min. The bacteria are made competent by taking up the bacterial pellet with a volume of TSB (miUeu LB + 100 g / 1 of PEG 4000, 5% of DMSO, 10 mM of MgCl ₂₎ 10 mM of MgSO ₄ ) corresponding to 1/10 of the volume of the medium of the initial culture. After incubation at 4 ° C for 30 to 60 minutes, 200 μl of bacteria are brought into contact with the Ugation products for 15 minutes on ice. After adding 200 μl of LB, the bacteria are incubated for 30 min at 37 ° C. and then spread on an LB + ampicillin medium.

9) The DNA separation and extraction are carried out as follows:

The DNA is separated according to their size by electrophoresis. To do this; different gels are used depending on the size of the fragments to be separated:

- 6%, 10% and 20% polyacrylamide precast polyacrylamide gels for the separation of small DNA fragments (75 to 500 bp)

- 1% agarose gel (Gibco BRL) in TBE buffer (Tris base 90mM; Borate 90mM; EDTA 2mM) for the separation of large DNA fragments (greater than 500 bpd)

- 2% NuSieve agarose gel (FMC Bioproducts) in TBE buffer for the separation of small fragments (less than 500 bpd). Any migration on agarose gel or on polyacrylamide gel is carried out in a TBE buffer and in the presence of a molecular weight marker (1Kb ladder, Gibco BRL). The DNA is mixed with 1/10 of the volume of the deposition blue (200 g / l of Ficoll, 0.5 g / l of bromophenol blue, 50 mM of EDTA) before being deposited on gel. After migration to 100 volts and staining with ethydium bromide (concentration 0.5 μg / ml of gel), the bands are visualized under the UV lamp.

The extraction of DNA from the band of an agarose gel is carried out by electroelution as follows: The piece of gel containing the DNA fragment is cut with a scalpel and placed in a dialysis rod closed by two forceps and containing 100 to 500μl of TBE. The whole is put in an electrophoresis tank where it undergoes an electric field of 100 Volts. The DNA, after being removed from the gel, is then purified by two phenol / chloroform extractions followed by two chloroform extractions, then precipitated in the presence of 0.3M sodium acetate and 2.5 volumes of absolute ethanol. After centrifugation (5 min at 14,000 rpm) the DNA pellet is dried and then taken up in 20 μl of water.

10) Fluorescent sequencing of plasmid DNAs.

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 the replication by Taq polymerase of the DNA to be sequenced. This reaction will give DNA fragments of different sizes, all terminated in 3 'with one of the 4 dideoxyribonucleotides. One μg of a plasmid and 4 picomoles of a primer are added to 9.5 μl of a "premix" supplied by AppUed Biosystems under the name of Prism. The final volume must be 20 μl to carry out a PCR for 25 cycles, decomposing into a denaturation step at 96 ° C for 30 seconds. A hybridization step at 50 ° C for 15 seconds and an elongation step at 60 °. C for 4 minutes. The DNA fragments, obtained after amplification, are purified on an exclusion column (Chromaspin-30 from Clontech); and are then dried with Speed Vac. The whole is taken up in 5 μl of a mixture formed of 24 μl of EDTA (50 mM) and 120 μl of deionized formamide. After denaturation at 96 ° C for 3 minutes, 3 to 5 μl are deposited on an electrophoresis gel. The different DNA fragments are separated according to their size and will pass successively in front of a laser reader of the Apparatus 370 DNA sequencer (Applied Biosystems) where the different fluorescences will be detected.

11) Preparation of plasmids from the brain bank (Clontech®).

The brain cDNA fusion bank is sold as bacteria. The latter contain a plasmid pGADIO containing an insert corresponding to a human brain cDNA. The cDNAs of this bank are formed using the oUgodT technique and the degenerate oUgonucleotide technique which allows to have the 5 'parts of the mRNAs which are difficult to obtain by the first technique. These cDNAs are cloned into the vector pGADIO at the EcoRI site.

After checking the title of the bank, 2 μl of bacteria from the brain fusion bank, put beforehand in 8 ml of LB, are spread non-confluently on a solid medium in order to keep the representativeness of this bank. We have thus spread 16 boxes of 770 cm containing a LU + ampicUine miUeu. The colonies that have appeared are taken up for each of the dishes with 30 ml of LB + ampicine Uquide. The suspensions obtained are then placed in an Erlenmeyer flask and incubated in a shaker 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 with a plasmid.

The yeasts previously cultivated in 100 ml of liquid medium are harvested after centrifugation at 3000 rpm for 3 minutes and suspended in 1 ml of sterile water. After centrifugation at 3000 m for 3 minutes, the cell pellet is resuspended in 1 ml of sterile water and then centrifuged again. This operation is repeated again in order to eliminate all traces of the culture medium. The yeasts are then taken up in 1 ml of the transformation solution I (0.1A LiAc, Tris-HCl pH 7.5 lOmM, EDTA ImM). then centrifuged at 3000 φm for 3 minutes. The cell pellet is taken up again in 1 ml of the transformation solution I. 50 μl of this yeast suspension are placed in the presence of 50 μg of salmon sperm DNA and from 1 to 5 μg of plasmid DNA. 300 μl of a transformation solution II (LiAc 0.1M, Tris-HCl pH 7.5 lOmM, EDTA ImM in PEG ₄₀₀₀ 40%) are then added, then the whole is incubated at 28 ° C for 30 minutes . A thermal shock is then applied to the transformation mixture in a water bath at 40 ° C for 15 minutes and then the whole is centrifuged at 15000 φm for 1 minute in order to collect the cell pellet. This pellet is taken up in 200 μl of water and then spread over a minimum agar medium which does not contain the amino acids corresponding to the markers provided by the transforming plasmid. The yeasts are then placed in culture for 72 hours at 28 ° C.

In the particular case of the transformation of yeast by the brain cDNA library, the procedure is as follows: The yeast used contains the plasmid pGAL4DB-CAPP coding for the C-terminal part of the APP fused to the DNA binding domain of GAL4. It is cultivated in 250 ml of medium minimum YNB-r-His + Lys + Ad + Leu at 28 ° C.

• Stirring up to a density of 10 ceUules / ml. The cells are harvested by centrifugation at 3000 àm for 10 minutes and taken up in 250ml of water. After a new centrifugation, the cell pellet is taken up in 100 ml of water and centrifuged again. The pellet is then taken up in 10 ml of the transformation solution I and incubated for 1 hour at 28 ° C. with shaking. After centrifugation, the cells are again taken up in 2.5 ml of the transformation solution I, 100 μl of the brain cDNA bank and 20 ml of the transformation solution II, then incubated for 1 hour at 28 ° C with stirring. A thermal shock is carried out on this transformation mixture at 42 ° C for 20 minutes. Centrifugation (3000 φm for 5 min) is repeated 3 times in succession, each time taking up the pellet with 10 ml of sterile water. The third time the pellet is taken up with 2.5 ml of PBS. Thus PEG toxic for cells has been eliminated. 2.4 ml of this suspension are used to inoculate 250 ml of minimum medium containing the amino acids His, Lys, Ad and cultured overnight in a shaker at 28 ° C. The remaining 100 μl of this suspension is used to verify the efficiency of the transformation; for this, dilutions of 10 ^" , 10 ^" and 10 of this suspension were made and spread on a minimum medium containing the amino acids His, Lys, Ad. After culture at 28 ° C. for 2 days, the colonies obtained were counted and the transformation rate is determined using the formula: "number of colonies x dilution factor". The overnight culture is centrifuged (3000 φm for 5 min) and washed with sterile water twice in succession. The pellet is then taken up in 2.5 ml of water. 2.4 ml, the volume of which is brought to 10 ml in sterile water, are used to inoculate 10 dishes of 435 cm containing YNB + Lys + Ad medium and incubated for 3 days. The remaining 100 μl are used to carry out the same operations as when determining the transformation rate, in order to determine the rate of amplification of the number of colonies during a culture night.

DNA (genomic and plasmid) is extracted from yeasts as follows :.

The value of an average loop of a yeast clone is put in 200 μl of a TELT solution (Triton XI 00 2%, SDS 1%, NaCl lOOmM, Tris pH8 lOmM, EDTA ImM), in the presence of 3g of biUes of glass 450 μm in diameter and 200 μl of phenol / chloroform. This mixture is vortexed for 15 minutes, then centrifuged for 2 minutes at 14000 φm. The supernatant is collected without removing the protein cake and the DNA contained in this phase is precipitated with 2.5 volumes of absolute ethanol. After centrifugation for 2 minutes at 14,000 φ, the DNA pellet is dried and taken up in 20 μl of TERNAse. This DNA solution, which corresponds to a mixture of genomic and plasmid DNA, is used directly to transform bacteria. Only plasmid DNA is capable of replicating in bacteria and can be analyzed by the miniprep technique.

13) βgalactosidase activity test.

A nitrocellulose sheet is previously deposited on the Petri dish containing the individualized yeast clones. Thanks to the phenomenon of addition, we will obtain a faithful image of the location of the clones. This sheet is then immersed in Uquide nitrogen for 30 seconds in order to burst the yeasts and thus to tiberate the βgalactosidase activity. After thawing, the nitroceUulose sheet is deposited, colonies upwards, in another Petri dish containing Whatman paper previously soaked with 1.5 ml of PBS solution (Na ₂ HPO4 60mM, NaH ₂ PO ₄ 40mM, KCl 10MM, MgSO ₄ ImM, pH7) and from 10 to 30 μl of X-Gal (5-bromo-4-chloro-3-indoyl-β-D-galactoside) at 50 mg / ml of N, N-dimethylformamide. The box is then placed in an oven at 37 ° C with the lid closed to prevent drying. The appearance time of the blue color can be very variable, from a few minutes to several hours. This test should always be done in the presence of a positive control whose interaction is known and quickly turns blue.

EXAMPLE 1 Construction of a vector allowing the expression of a fusion protein between the C-terminal part of the precursor of the amyloid peptide (CAPP) and the DNA binding domain of GAL4

Screening of a library using the double hybrid system requires that the C-terminal region of the APP (CAPP) be fused to the DNA binding domain of the GAL4 transactivating protein. The expression of this fusion protein is achieved using the vector pGBTIO (see materials and methods), in which we have introduced, in the same reading frame as the sequence corresponding to the Uaison domain to the DNA of GAL4 (GAL4DB), the sequence coding for CAPP appearing in the sequence presented in SEQ ID No. 1.

The 138 bpd DNA fragment corresponding to the last 46 amino acids of the APP was obtained by PCR from the Ogonucleotides (SEQ ID N ° 3 and N ° 4) which also allowed us to introduce the EcoRI and Dirty at the ends of the sequence. The PCR fragment was introduced between the EcoRI and SalI sites of the multisite for cloning the plasmid pGBTIO downstream of the sequence corresponding to GAL4DB to give the vector pGAL4DB-CAPP (FIG. 2). The construction was verified by DNA sequencing. This verification allowed us to show that this fragment did not present mutations generated during the PCR reaction and that it was fused in the same open reading phase as that of the fragment corresponding to GAL4DB.

EXAMPLE 2: SCREENING OF THE BRAIN FUSION BANK.

The screening of a fusion bank makes it possible to identify clones producing proteins fused to the transactivating domain of GAL4, which can interact with our protein of interest. This interaction makes it possible to reconstitute a transactivator which will then be capable of inducing the expression of the reporter genes His3 and LacZ in the YCM strain.

To carry out this screening, we chose a fusion library made from cDNA from the human brain. As this library was supplied to us in the form of bacteria, the plasmid DNA of the library was first purified.

2.1) Preparation of the plasmid DNA of a fusion library.

The plasmid DNA from the brain cDNA library was extracted according to the Clontech protocol (see materials and methods, § 11). During this preparation, it was important to preserve the representativeness of the library, that is to say, to keep the number of independent plasmids which constitute it and which are 1.2.10 ⁶ plasmids. In order to protect us from the loss of plasmids from the library during this preparation, the batch of plasmid DNA that we have assembled was obtained from a number of isolated bacterial colonies corresponding to slightly more than twice the representativeness of the bank, ie 4.10 ⁶ colonies.

2.2) Transformation by brain bank and selection by the βgalactosidase activity test.

During screening, it is necessary to preserve the probability that each plasmid independent of the fusion library is present in at least one yeast at the same time as the plasmid GAL4DB-CAPP. To preserve this probability, it is important to have a good yeast transformation efficiency. For this we have chosen a yeast transformation protocol giving an efficiency of 10 ⁵ ceUules transformed per μg of DNA. In addition, since the cotransformation of the yeast by two different plasmids reduces this efficiency, we preferred to use a yeast previously transformed by the plasmid pGAL4DB-CAPP. This YCM-CAPP strain of His-, Lys-, Leu- phenotype was transformed with lOOμg of plasmid DNA from the fusion bank. This quantity of DNA allowed us to obtain after estimation (see materials and methods) 10 transformed cells, which corresponds to a number slightly higher than the number of independent plasmids that constitute the bank. From this result we can think that almost all of the plasmids from the bank were used to transform yeasts. The selection of the transformed cells, capable of reconstituting a functional GAL4 transactivator, was made on a YNB + Lys + Ad medium.

At the end of this selection 1000 clones with a His + phenotype were obtained. On these transformants, a βgalactosidase activity test was carried out in order to assess the number of clones obtained by the expression of the other reporter gene, LacZ. 68 of the 1000 clones obtained exhibited the double His + phenotype, βGal + which may correspond to a protein-protein interaction.

EXAMPLE 3: ISOLATION OF BANK PLASMIDS

In order to identify the proteins that can interact with CAPP, we extracted the plasmids from the fusion bank contained in the yeasts selected during the double hybrid screening. To be able to obtain large quantities, this isolation requires a transformation of E. coli beforehand with a DNA extract of the positive yeast strains. As the bank plasmid contained in this extract is a yeast / E. coli shuttle plasmid, it can easily repute in bacteria. In order to avoid isolating the plasmid GAL4 DB - CAPP which is also present in yeast, we have worked on strains which do not have it.

The plasmid DNAs of the bacterial colonies obtained after transformation with yeast DNA extracts were analyzed by digestion with restriction enzymes and separation of the DNA fragments on agarose gel. We obtained 3 different restriction profiles on the 5 clones analyzed (7D, 3E, 9A, 3H, 3G) (fig 2). These results showed that 2 identical plasmids are at least represented in 2 different yeast clones, the 7D clones, as well as the 3H and 3G clones. The DNA of clone 3E is obtained from strains of the His + and βGAL + phenotype.

EXAMPLE 4: DETERMINATION OF THE SEQUENCE OF THE INSERTS OF THE IDENTIFIED PLASMIDS.

The sequencing was carried out using the oligonucleotide (SEQ ID No. 5) complementary to the GAL4TA region near the insertion site of the brain cDNA library, 52 bpd from the EcoRI site. The comparison of the 3 sequences with the sequences contained in the GENBank and EMBL (European Molecular Biology Lab) databases showed that the sequences of the cDNAs which were in the plasmids derived from strains 9A and 3H show 87% homology at the nucleic level with the mutant gene coding for the protein FE65, they are represented in SEQ ID No. 2, and that the sequence of the plasmid derived from the strain 7D has 60% homology with this same gene (see FIG. 3). Analysis of the sequence of the plasmids from strains 9A and 3H indicates that these contain two overlapping regions corresponding to the same mRNA. LIST OF SEQUENCES

(1) GENERAL INFORMATION:

(ι) DEPOSITOR:

(A) NAME: RHONE POULENC RORER S.A.

(B) STREET: 20, Avenue Raymond Aron

(C) CITY: ANTONY (E) COUNTRY: FRANCE

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(G) TELEPHONE: 40.91.69.22

(H) FAX: (1) 40.91.72.96 (n) TITLE OF L ¹ INVENTION:

(ni) NUMBER OF SEQUENCES: 5

(îv) COMPUTER-DETACHABLE FORM: (A) TYPE OF SUPPORT: Tape

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patentln Release # 1.0, Version # 1.30 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(î) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1500 (B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(n) TYPE OF MOLECULE: cDNA

(Xl) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1

(2) INFORMATION FOR SEQ ID NO: 2:

(î) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 1275

(B) TYPE: nucleotide (C) NUMBER OF STRANDS: Single

(D) CONFIGURATION: linear (il) TYPE OF MOLECULE: cDNA

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:

CGG GAG GAG TCC CAG CTC ACC TGG ACA GGT TTT GCT CAT GGA GAA GGC 48 Arg Glu Glu Ser Gin Leu Thr Trp Thr Gly Phe Ala His Gly Glu Gly 1 5 10 15

TTT GAG GAT GGA GAA TTT TGG AAG GAT GAA CCC AGT GAT GAG GCC CCA 96 Phe Glu Asp Gly Glu Phe Trp Lys Asp Glu Pro Ser Asp Glu Ala Pro 20 25 30

ATG GAG CTG GGA CTG AAG GAA CCT GAG GAG GGG ACG TTG ACC TTC CCA 144 Met Glu Leu Gly Leu Lys Glu Pro Glu Glu Gly Thr Leu Thr Phe Pro 35 40 45

GCT CAG AGC CTC AGC CCA GAG CCG TTG CCC CAA GAG GAG GAG AAG CTT 192 Ala Gin Ser Leu Ser Pro Glu Pro Leu Pro Gin Glu Glu Glu Lys Leu 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

65 70 75 80

CTA GGC TGG GTA GAG ATG ACC GAG GAG GAG CTG GCC CCT GGA CGC AGC 288

Leu Gly Trp Val Glu Met Thr Glu Glu Glu Leu Ala Pro Gly Arg Ser

85 90 95 AGT GTG GCA GTC AAC AAT TGC ATC CGT CAG CTC TCT TAC CAC AAA AAC 336

Ser Val Ala Val Asn Asn Cys Ile Arg Gin Leu Ser Tyr His Lys Asn

100 105 110

AAC CTG CAT GAC CCC ATG TCT GGG GGC TGG GGG GAA GGA AAG GAT CTG 384 Asn Leu His Asp Pro Met Ser Gly Gly Trp Gly Glu Gly Lys Asp Leu

115 120 125

CTA CTG CAG CTG GAG GAT GAG ACA CTA AAG CTA GTG GAG CCA CAG AGC 432

Leu Leu Gin Leu Glu Asp Glu Thr Leu Lys Leu Val Glu Pro Gin Ser 130 135 140

CAG GCA CTG CTG CAC GCC CAA CCC ATC ATC AGC ATC CGC GTG TGG GGC 480

Gin Ala Leu Leu His Ala Gin Pro Ile Ile Ser Ile Arg Val Trp Gly

145 150 155 160

GTC GGG CGG GAC AGT GGA AGA GAG AGG GAC TTT GCC TAC GTA GCT CGT 528 Val Gly Arg Asp Ser Gly Arg Glu Arg Asp Phe Ala Tyr Val Ala Arg 165 170 175 GAT AAG CTG ACC CAG ATG CTC AAG TGC CAC GTG TTT CGC TGT GAG GCA 576 Asp Lys Leu Thr Gin Met Leu Lys Cys His Val Phe Arg Cys Glu Ala 180 185 190

CCT GCC AAG AAC ATC GCC ACC AGC CTG CAT GAG ATC TGC TCT AAG ATC 624 Pro Ala Lys Asn Ile Ala Thr Ser Leu His Glu Ile Cys Ser Lys Ile 195 200 205

ATG GCC GAA CGG GGT AAT GCC CGC TGC TTG GTA AAT GGA CTC TCC CTG 672 Met Ala Glu Arg Gly Asn Ala Arg Cys Leu Val Asn Gly Leu Ser Leu 210 215 220

GAC CAC TCT AAA CTT GTG GAT GTC CCT TTC CAA GTG GAA TTC CCA GCG 720 Asp His Ser Lys Leu Val Asp Val Pro Phe Gin Val Glu Phe Pro Ala 225 230 235 240

CCT AAG AAT GAG TTG GTC CAG AAG TTC CAA GTC TAT TAC CTG GGG AAT 768 Pro Lys Asn Glu Leu Val Gin Lys Phe Gin Val Tyr Tyr Leu Gly Asn 245 250 255 GTA CCT GTT GCT AAA CCT GTT GGG GTA GAT GTG ATT AAT GGG GCC CTC 816 Val Pro Val Ala Lys Pro Val Gly Val Asp Val Ile Asn Gly Ala Leu 260 265 270 GAG TCA GTC CTG TCC TCC AGC AGC CGT GAA CAA TGG ACC CCA AGT CAT 864 Glu Ser Val Leu Ser Ser Ser Ser Arg Glu Gin Trp Thr Pro Ser His 275 280 285

GTC AGT GTG GCC CCT GCT ACC CTC ACC ATC TTG CAC CAG CAG ACA GAG 912 Val Ser Val Ala Pro Ala Thr Leu Thr Ile Leu His Gin Gin Thr Glu 290 295 300

GCA GTG CTG GGA GAG TGT CGG GTG CGT TTC CTC TCC TTC CTG GCC GTG 960 Ala Val Leu Gly Glu Cys Arg Val Arg Phe Leu Ser Phe Leu Ala Val 305 310 315 320

GGC AGA GAT GTC CAC ACG TTT GCA TTC ATC ATG GCT GCC GGC CCA GCC 1008 Gly Arg Asp Val His Thr Phe Ala Phe Ile Met Ala Ala Gly Pro Ala 325 330 335

TCC TTC TGC TGT CAC ATG TTC TGG TGC GAG CCC AAT GCT GCC AGC CTC 1056 Ser Phe Cys Cys His Met Phe Trp Cys Glu Pro Asn Ala Ala Ser Leu 340 345 350 TCA GAG GCT GTG CAG GCT GCG TGC ATG CTT CGC TAC CAG AAG TGT CTG 1104 Ser Glu Ala Val Gin Ala Ala Cys Met Leu Arg Tyr Gin Lys Cys Leu 355 360 365

GAT GCC CGT TCC CAG GCC TCC ACC TCC TGC CTC CCA GCA CCC CCT GCT 1152 Asp Ala Arg Ser Gin Ala Ser Thr Ser Cys Leu Pro Ala Pro Pro Ala 370 375 380

GAG TCT GTG GCA CGG GGT GTA GGG TGG ACT GTC CGC AGG GGT GTT CAG 1200 Glu Ser Val Ala Arg Gly Val Gly Trp Thr Val Arg Arg Gly Val Gin 385 390 395 400

TCG CTG TGG GGC TCC CTG AAG CCC AAA CGG GTG GGG GGC CAT ACC CCA 1248 Ser Leu Trp Gly Ser Leu Lys Pro Lys Arg Val Gly Gly His Thr Pro 405 410 415

TGA AGA AGC CCC ACC TTC CCT CCA CCT 1275 * Arg Ser Pro Thr Phe Pro Pro Pro 420 425

(2) INFORMATION FOR SEQ ID NO: 3:

(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 27 (B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: CAAGTCGACC TAGTTCTGCA TCTGCTC 27

(2) INFORMATION FOR SEQ ID NO: 4:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH

(B) TYPE: nucleotide (C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4:

CAAGAATTCA AGAAACAGTA CACATCC

(2) INFORMATION FOR SEQ ID NO: 5:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 23

(B) TYPE: nucleotide (C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5:

CTATTCGATG ATGAAGATAC CCC 23

Claims

1. Peptide characterized in that it is capable of interfering at the level of the interaction of the protein FE65 or one of its homologous forms with the cytoplasmic region of APP.

2. Peptide according to claim 1 characterized in that the interference results in a slowing down, an inhibition or a 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 thereof.

5. Peptide capable of competing with a peptide according to any one of claims 1 to 4 at the level of the interaction domain between the protein FE65 or one of its homologous forms and the cytoplasmic region of APP.

6. A non-peptide or non-exclusively peptide compound capable of modulating the interaction between the FE65 protein and the cytoplasmic region of the APP obtained by reproduction of the active motifs of the peptides according to claims 1 to 5 by non-peptide or non-exclusively peptide structures .

7. Nucleotide sequence coding for a peptide as defined in claims 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 No. 2 or a sequence derived therefrom.

9. The sequence of claim 7 or 8 introduced into a viral or non-viral vector.

10. Antisense oligonucleotide of a sequence according to claim 7 or 8.

11. Anticoφs or fragment of anticoφs characterized in that it is directed against 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 principle at least one peptide according to one of claims 1 to 6 or an anticoφs or fragment of anticoφs 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 eUe 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 U 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 protein FE65 and the cytoplasmic region of APP.

17. Pharmaceutical composition according to claim 16 or 17 intended to interfere with the interaction between the protein FE65 and the cytoplasmic region of APP.

18. Pharmaceutical composition according to claim 16 intended for the treatment of neurodegenerative diseases.

19. Use of an anticoφs or fragment of anticoφs according to claim 11 and / or a nucleotide sequence according to one of claims 7 or 8 for the typing of neurodegenerative diseases.

20. A method of preparing 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 is cultured under conditions of expression of said sequence and recover the peptide produced.

21. Defective recombinant virus comprising a heterologous nucleic sequence coding for a polypeptide according to one of claims 1 to 5.