WO2001009613A1

WO2001009613A1 - Method for screening molecules for the treatment of huntington disease

Info

Publication number: WO2001009613A1
Application number: PCT/FR2000/002036
Authority: WO
Inventors: Christian Neri
Original assignee: Fondation Jean Dausset-Ceph
Priority date: 1999-07-29
Filing date: 2000-07-13
Publication date: 2001-02-08
Also published as: CA2380519A1; FR2797055A1; FR2797055B1; EP1200834A1

Abstract

The invention concerns a method for screening molecules for the treatment of Huntington disease and a method for identifying therapeutic targets for said disease. The methods are based on the indentification of molecules which are capable of modifying the interaction normal htt/ CA 150.

Description

METHOD FOR SCREENING MOLECULES FOR THE TREATMENT OF HUNTINGTON MALADD2

The subject of the present invention is a method for screening molecules intended for the treatment of Huntington's disease as well as the use of molecules thus demonstrated for the preparation of a medicament intended for the curative or preventive treatment of this disease. It also relates to a method of identifying therapeutic targets for Huntington's disease and the use of nucleotide sequences corresponding to genes involved in the development of this disease in order to treat it.

Huntington's Disease (Huntington Disease or HD) is an autosomal dominant neurodegenerative disease with polyglutamine expansion (polyQ) whose gene codes for huntingtin (htt), an unknown function protein expressed in the brain and several other tissues. The expansion of polyQ (following a dynamic mutation-or expansion-CAG in the htt gene) is located in the N-terminal part of the htt, a region that is conserved and functionally important for the appearance of HD. The normal htt contains a normal number of glutamine repeats (Gins) (typically: 15 to 20 Gins, mainly: less than 39 Gins). The mutated htt contains an abnormally high number of glutamine repeats (typically the pathological threshold is around 39 Gins, the expansion of polyglns can for example reach up to 180 Gins in certain cases) which is associated with the appearance of disease (Koshy and Zoghbi, 1997). Htt is an important protein for the genesis and survival of the neuron (Zeitlin et al., 1995; White et al., 1997). In the normal state, this protein is mainly located in the cytoplasm of neurons where it is for example associated with microtubules (DiFiglia et al., 1997). Normal htt has also been detected in the nucleus, in mouse neuroblastoma cells (de Rooij, 1996), and in the post-mortem human brain (Hoogeven et al., 1993).

On the tissue level, HD is characterized in the first place by a progressive and selective apoptosis of the striatal neurons; at later stages, HD affects other regions of the brain such as the cortex (Koshy & Zoghbi, 1997). The molecular bases of this selectivity are not known.

As normal and mutated forms of htt are expressed at similar levels, the cause of HD is to be sought in biochemical disturbances in the signaling pathways involved in the genesis, functioning, and survival of the neurons affected in HD.

Furthermore, there is an inverse correlation between the age of onset and the severity of HD and / or the number of glutamine repeats in the htt (Koshy and Zoghbi, 1997), which indicates that the severity of the disturbance of these biochemical functions is modulated by the length of the polygln domain.

There is no treatment for this seriously disabling disease (6000 people in France, apparent prevalence close to 1 / 10,000). The identification of the biochemical factors involved in HD is necessary in order to identify therapeutic targets for this disease, and to proceed to the search for therapeutic methods and active compounds making it possible to restore normal functioning of the intracellular signaling pathways important for survival. and the proper functioning of the affected neurons in HD. In patients, nuclear aggregates (sometimes perinuclear) composed of N-terminal fragments of htt and ubiquitin have been demonstrated (DiFiglia et al, 1997). The fact that aggregates were detected before the appearance of apoptosis in in vitro cell models and in transgenic mouse models initially led to the hypothesis that nuclear aggregates could be at the origin of the disease (Mangiarini et al., 1996; Davies et al., 1997; Ross, 1997). However, the most recent results strongly support the hypothesis of an effect of the free forms of mutated htt as a fundamental pathophysiological mechanism of HD (Sisodia, 1998). Several studies (on post-mortem brains, primary cultures of rat striatal neurons, other cellular models in vitro, and models in mice) indeed indicate that there does not exist a good correlation between the formation of aggregates and apoptosis of target neurons (Bâtes et al., 1998; Lunkes and Mandel, 1998; Saudou et al., 1998; Sisodia, 1998; Hackam et al., 1999; Schilling et al., 1999). These aggregates are moreover likely to fortuitously protect the neuron against an excess of apoptosis caused by the non-aggregated mutated htt (Saudou et al., 1998). On the other hand, the nuclear localization of “free” N-terminal fragments of the mutated htt is necessary for the induction of neuronal apoptosis (Saudou et al., 1998).

The nucleus could therefore be the privileged place of the pathophysiological mechanisms of HD, in particular in terms of dysregulation of transcription and inadequate expression of certain genes and proteins.

Another important aspect of the pathophysiology of HD lies in the fact that neuronal dysfunction before apoptosis is highly likely to be at the origin, in whole or in part, of this disease. In transgenic models of HD in mice, it has indeed been observed that behavioral symptoms in transgenic mice (expression of an N-terminal fragment of the mutated htt corresponding to exon 1 of the gene for this protein) appear before detecting apoptosis of neurons (Bâtes et al., 1998).

Furthermore, the expansion of polyglns probably induces a change in conformation of the N-terminal fragments of htt, the pathological consequences of which would go hand in hand with the progressive proteolytic degradation of htt (initially by caspases like caspase-3, then by other unidentified proteases) and the genesis of shorter and shorter N-terminal fragments comprising for example amino acids 1-500, 1-300, then 1-150, and 1-100 (Goldberg et al., 1996; Lunkes and Mandel, 1998; Martindale et al., 1998; Sharp et al., 1998; Schilling et al., 1999), and their passive diffusion into the nucleus through nuclear pores (Hackam et al., 1999) . Indeed, the first 50 to 200 amino acids are sufficient to induce cellular or behavioral phenotypes similar to those observed in HD in transgenic models of mice (Mangianiri et al., 1996; Bâtes et al., 1998), of Drosophila (Jackson and al., 1998), and nematode C. elegans (Faber et al., 1999; Néri et al., 1998).

The expansion of polyQ (through the change in conformation mentioned above) would also modify the accessibility of huntingtin partner proteins (PPH) to their binding sites in the htt (apart from polyQ domain), resulting in a decrease or an increase in the binding of mutated htt with PPH belonging to intracellular signaling pathways essential for the functioning and survival of the neuron. The potential role of abnormal protein-protein interactions in HD has been illustrated by the identification in double-hybrid screening (Fields and Song, 1989) of several PPHs which bind to the N-terminal region of htt (amino acids 1 to 500). The majority of these PPHs seem to bind to htt outside of the polygln domain. In the case where these PPHs have been identified by screening of non-human cDNA libraries (for example, rat or mouse cDNAs), it has been shown that the human counterparts of these PPHs do indeed bind to htt in vitro, and that they co-localize with htt in extracts from the human brain. In general, there is no systematic correlation between the expression profile of PPH identified to date and the sites of neuronal loss observed in HD. These are essentially the following PPHs:

- GAPDH (Burke et al., 1996; increased invariant link with mutated htt): enzyme involved in glycolysis and energy metabolism.

- HTP2 (Kalchman et al., 1996; invariant link with the mutated htt): Conjugation enzyme with ubiquitin. - Cystathionine β-synthase or CBS (Boutell et al., 1998; invariant link with mutated htt): key enzyme for the synthesis of cysteine from methionine. The absence of CBS is associated with the accumulation of the CBS substrate which is excitotoxic (homocysteine), and with a rare human metabolic disease (homocystinuria). The role of a similar mechanism in HD has not been established.

- HAP1 (Li et al., 1995 and W0 97/17443; increased binding with mutated htt) and HDPl (Kalchman et al, 1997; Wanker et al. 1997; decreased binding with mutated htt): proteins of unknown function associated with the cytoskeleton whose binding sites seem to lie between amino acids 171 and 500. HAP1 interacts with several proteins, in particular the pl50 ^G '" ^erf subunit of dynactin (Engelender et al., 1997), which confirms the role of HAP1 in the formation and activity of the cytoskeleton and the trafficking of intracellular vesicles. - HYPA and HYPB (Faber et al., 1998; increased binding with mutated htt): proteins with WW domain of unknown function, binding at the level of the two polyprolines immediately at the C-terminal of the polyQ domain.

- SH3GL3 (Sittler et al., 1998: increased binding with mutated htt): protein of unknown function with SH3 domain belonging to the Grb2 family, the members of this family being associated with synaptosomes, binding at the level of the two polyprolines immediately C- terminale à la polyglns. Promoter role of SH3GL3 in the formation of aggregates.

It is important to note three points:

1) Several PPHs which exhibit an increase in binding to the mutated htt (HAP1, SH3GL3, HYP series) are associated with the formation of nuclear aggregates such as SH3GL3 which has a promoter role on their formation (Sittler et al., 1998), or HIP2 which is directly associated with the kinetics of degradation or accumulation of intracellular proteins. As aggregates are not a sine qua non cause of apoptosis (Saudou et al., 1998; Sisodia, 1998), the search for a blockage of an abnormally increased and "neurotoxic" link between the mutated htt and a PPH by an active compound (as described by C. A Ross et al., Ingtuntingtin-associated protein ', WO97 / 17443) is in fact likely not to constitute a good therapeutic option. In addition, these PPHs a priori show moderate variations in interaction (generally by a factor of 2 to 5) for mutated htt (40-60 Gins) compared to normal htt (20 Gins). By virtue of the state of the art in the field of small molecules, it is difficult to envisage the specific “beneficial” blocking of a moderately increased link between the mutated htt and a PPH, in the absence of a side effect "Inappropriate" blocking of the link between normal htt and this same PPH.

2) HIPl is the only PPH which has so far shown a loss of connection with the mutated htt. HIP1 interacts approximately 10 times less well with mutated htt comprising 125 Gins than with normal htt comprising 25 Gins (Kalchman et al., 1997 and WO 97/18825). This protein is specifically expressed in the brain, more specifically in the cortex. Its function is unknown: the homology of 20-40% with Sla2p suggests a role in cytoskeleton formation, and the presence of a “Leucine zipper” motif suggests involvement in several protein-protein interactions. The role of HIP1 in the formation of the cytoskeleton is supported by the fact that this protein is associated with cell membranes (cell debris, nucleus, synaptosomes, mitochondria, microsomes and plasma membranes), a common characteristic of all cytoskeleton proteins such as Tartine. Since HIPl is not linked to the formation of aggregates, it is an interesting therapeutic target. However, the fact that it is a cytoskeleton protein does not fall within the framework of the latest data in the scientific literature which indicates that the nuclear localization of the mutated htt is necessary for the establishment of the pathophysiological mechanisms of HD (Saudou et al. 1998; Sisodia, 1998). 3) The influence of the length of the N-terminal fragment (in particular those likely to pass into the nucleus) on the binding of htt to PPH has not been described previously.

In this context, the inventors provide the following new elements: identification of a new WW domain PPH whose human counterpart is a nuclear transcription factor containing a polymorphic domain (repetition of QA or Glu-Ala dipeptides), and identification of biochemical conditions (size of the fragments of the htt) which go hand in hand with notable differences in the binding of this PPH with the mutated htt and the normal htt in the nucleus. The therapeutic targets and the strong differences identified by the inventors are compatible with the concept of therapeutic interventions which aim to act on the pathological effects of certain forms of the mutated htt, without side effects on the normal physiological functions of certain forms of the htt normal. Double-hybrid screening for htt partners is a particularly interesting entry point into the biology of affected neurons in HD. The ultimate proof that PPH is actually involved in neuronal impairments comes from additional in vitro and in vivo experiments. For example, it is possible to prove in vivo in a whole organism 1) that the candidate target is actually involved in the disease phenotype (loss of function or apoptosis) reproduced in the model organism used, 2) that it is possible to modulate its biological activity, and 3) that this modulation can restore normal functioning of neurons rendered deficient by the mutated htt. This so-called “cellular” validation (establishment of a direct link between a biochemical phenotype and a cellular phenotype) can be carried out in an entire organism and in a relatively short time thanks to the use of the nematode Caenorhabditis elegans. Thus, it is possible to identify the "genetic pathways" involved in a neuronal deficiency phenotype through the search for suppressor mutants. C. elegans also makes it possible to study PPH or “physical pathways” (protein-protein interactions) of interest and of unknown function which were initially identified by double-hybrid screening in yeast, by making it possible to study the profiles of expression and the mutant phenotypes for the homologs of these PPHs in C. elegans.

In terms of prospective therapeutics in neurobiology, C. elegans has the following advantages: well-characterized nervous system, good functional homology with humans in terms of neuronal physiology, many normal neurological phenotypes and mutants available for analysis. Compared to other simple model organisms such as Drosophila (Jackson et al., 1998), C. elegans also has the following advantages: completely sequenced genome, faster mutagenesis, transparent organism allowing visualization of all neurons and a analysis of protein expression in living animals, short reproduction cycle (a few days), knowledge of complete cell phylogeny, ease of use (culture on solid or liquid agar, resistance of larvae to freezing, and possibility high throughput screening (e.g. 96-well microplates). Due to the strong conservation of several intracellular signaling pathways between the nematode and other species including humans, especially in terms of neuronal physiology (Bargman, 1998; Ruvkun and Hobert, 1998; The C. elegans Sequencing consortium, 1998 ), the study of human disease mechanisms in C. elegans is recognized as a powerful study tool for the identification and validation (at the cellular level, and before anatomo-functional pharmacological study in more complex models like the mouse or primate) of therapeutic targets. More particularly, the inventors set out to study the interactions between a human disease protein, htt, and all of the proteins listed in C. elegans, using the double-hybrid screening technique (Fields and Song, 1987) so as to reconstruct, at least in part, the diagram of the signaling pathways which are involved in the normal functioning of htt before polyQ expansion, and in the pathological functioning of htt after polyQ expansion. Following the double-hybrid screening, the analysis of homologies between the gene of interest identified in C. elegans and its human counterparts makes it possible to effectively predict the nature of the pathological mechanisms at play in HD and to define the first bases of 'a therapeutic intervention, to which the inventors have also focused.

The data obtained by the inventors therefore relate to the identification of phenomena of variation in interaction between a protein of C. elegans which turns out to be a protein with a WW domain of 946 amino acids and certain fragments of normal or mutated htt (loss of 'interaction). Although the function of ZKl 127.9 is unknown in the nematode, this protein has characteristics which strongly suggest that the activity of regulating the transcription of the genes of the human homolog of ZKl 127.9 (CA150) is modulated by N- fragments. htt terminals whose molecular weight is compatible with a passage in the nucleus of neuronal cells through nuclear pores.

Indeed, the N-terminal fragments of the htt used by the inventors, in particular httl52, have the capacity, due to their molecular weights (approximately 10-70 kDa), to penetrate into the nucleus of neurons by nuclear pores, no doubt passively (Hackam et al., 1998).

In addition, ZKl 127.9 187-758 has a loss of interaction of 92

% with the mutated form httl52Q128 compared to httl52Q15. When the htt size is 546 amino acids, the average interaction loss of

ZK1127.9 187-758 for htt546Ql28 in comparison to htt546Q15 is 56%. The phenomenon of loss of interaction between ZK1127.9 187-758 and htt is all the more marked as the size of the N-terminal fragments of the mutated htt is reduced. This loss of interaction is likely to be complete for any fragment of the htt containing less than 546 amino acids and more than 90 amino acids. Indeed, ZKl 127.9_187-758 is not able to interact with htt90Q15, which indicates the region 90-546 of the htt contains the binding sites for ZKl 127.9. The loss of interaction between ZK1127.9_187-758 and htt is practically complete for httl52Q128. The demonstration of a loss of interaction between mutated htt and WW domain proteins, and the fact that this loss is dependent on the length of the N-terminal fragment of the htt are new facts, which signal a potentially new mechanism important of neuronal apoptosis or neuronal dysfunction in HD, and underline the great potential value of the couple htt-CA150 as a therapeutic target. Furthermore, ZKl 127.9 has significant sequence homology with the human nuclear transcription factor CA150, a factor whose gene appears to be expressed in the human brain. This strongly suggests that certain forms of the mutated htt are unable to bind CA150 properly in the brains of patients. This also suggests that several human genes are not correctly expressed in HD due to a deficiency in the mechanisms of transcription regulation under the control of the htt-CA150 couple.

The expression profiles of human ESTs corresponding to the htt and to CA150 indicate that these two proteins are expressed in the brain. Furthermore, the fact that CA150 is a nuclear protein, that the size of the fragments of the htt tested by the inventors is compatible with a passage in the nucleus, and that the htt is moreover detected in the nucleus of neuronal cells, indicates that CA150 and htt most likely co-locate in the nucleus of brain neurons. With regard to the interactions 'htt- ZKl 127.9' in vitro: After expression and purification of GST :: htt fusion proteins and of ZK1127.9 :: epitopeHA in E. Coli (vector pGEX, and vector pGEX- epitopeHA such as modified by the inventors), fixing of the htt on sepharose beads (GST affinity resin, Stratagene), and incubation with ZKl 127.9 :: HA, the retention of ZKl 127.9 :: HA by normal and mutated htt was tested by western blot using an anti-HA antibody.

As regards the htt-CA150 interactions in vitro: after expression and purification of GST fusion proteins:: htt and of CA150 (fragment or whole protein) in E. coli (vector pGEX and vector pGEX- HA epitope as modified by the Inventors), fixing of the htt on sepharose beads (GST affinity resin, Stratagene), and incubation with CA150:: HA, the retention of CA150 by the normal htt was tested by western blot using anti- HA or anti-CA150 antibodies. But also, after expression and purification of CA150 (source of the complete cDNA: IMAGE network) in E. Coli, coupling of CA150 on sepharose beads and incubation with protein extracts from normal lymphoblastoid lines or from patients suffering from HD, and from post-mortem brains (healthy or sick individuals), the retention of htt by CA150 can be tested by western blot using anti-htt antibodies whose epitopes are located at the level of the first 600 amino acids of htt . The co-localization of CA150 with htt in these human tissues can be verified by conventional subcellular fractionation and western blot techniques as previously described (Sittler et al., 1998). In addition, human-CA150 htt interactions can be tested directly in a double-hybrid in yeast.

As regards the biochemical properties of CA150, during the CA150 production experiments:: HA in E. coli and the control of production by western blot using an anti-HA antibody or an antiCA150 antibody, the inventors have demonstrated a non-aggregated form of CA150 migrating on gel substantially at the expected size (approximately 92 kDA), and aggregated forms of CA150 migrating largely above the expected size. The aggregation properties of CA150 are most certainly to be attributed to the N-terminal repeat region (QA) 38, which, as in the case of polyglutamines or polyalanines in other human proteins, prove capable of promoting the aggregation of the carrier protein with itself, or even with other proteins. The aggregation properties of CA150 demonstrated by the inventors are capable of participating in the mechanisms of Huntington's disease. Regarding the function of ZKl 127.9 in C. elegans: the data described make it possible to study the role of ZKl 127.9 in C. elegans using standard transgenesis and mutagenesis techniques in C. elegans: profile of expression of ZKl 127.9 in C. elegans after expression in transgenesis of fusion proteins ZKl 127.9:: GFP (typically 1-2 kb of sequence upstream from exon 1 plus the first intron are sufficient for the expression of a protein in C. elegans), and analysis of the phenotype of non-lethal mutants for the gene of ZKl 127.9 (selection of the mutants by PCR screening of DNA pools corresponding to a library of mutants induced by exposure to TMP and UV; TMP mutagenesis / UV generating a majority of small deletions, it is particularly suitable for PCR screening).

Regarding cell validation of therapeutic targets in a living animal: with confirmation of loss of interaction between mutated htt and CA150 and on the basis of information collected on the function of ZKl 127.9, the binding sites between CA150 and Normal htt can be identified (for example by deletion mapping) in order to develop in vitro and in vivo tests which allow the identification of active compounds (peptides or organic compounds) capable of reconstituting the normal activity of the couple htt- CA150. The in vivo efficacy of such compounds can be directly tested in HD animal models which allow pharmacology studies in terms of toxicity, pharmacokinetics, effective doses (for example in primates, cf. Palfi et al., 1998).

CA150 is a modulator factor of nuclear transcription which interacts with RNA polymerase type II (Shune et al., 1997), and carries a repeating zone (rich in Alanine and Glutamine) which is present in several other modulating factors of the transcription as GAL11 or SSN6 in yeast, or Zeste in Drosophila. Using nucleotide primers at the boundaries of the nucleotide region corresponding to the domain (QA) 60 and after conventional analysis of the PCR products on polyacrylamide gel and autoradiography, the inventors have demonstrated that the domain (QA) 60 of CA150 is polymorphic (4 alleles between 57 and 62 QA, 30% heterozygosity) in the normal population after testing 20 healthy individuals (10 father-mother couples; CEPH reference families of American origin number 102, 1332, 1331, 1340, 1347, 1362, 1413, 1416, 1423, 884). Due to the polymorphism and the position of the polyQA domain (between 2 WW domains), these data suggest that the polymorphism of QA repeats in CA150 may be involved in the onset and progression of HD in patients, and may modulate the CA150 physiological activity, in particular its link with htt, all the more so since the QA repetitions in CA150 are also involved in CA150 aggregation phenomena as observed by the Inventors. Analysis of the homologies of CA150 with the human TSEs available in Genbank and in dbEST has made it possible to establish that the CA150 gene is expressed in several human tissues including the brain (uterus, tonsils, breast, muscle, kidney, lung, parathyroid , testicle, heart, whole embryo, colon, ear, blood). 80 human ESTs (representing 71 cDNA clones) correspond to the CA150 gene (chromosome location chromosome 5) according to Unigene analysis

(http://www.ncbi.nlm.nih.gov/cgi-bin/Unigene), of which 4 ESTs correspond to 4 brain cDNA clones (clone IMAGE 38349, and clones GEN-158H06, GEN-121H03, and HFBCA63). Four other human brain cDNA clones corresponding to CA150 (Genbank accession numbers M78358, R87450, AI216964, and HUM121HO3A) could be detected by the inventors in the databases.

These ESTs coming from sequences obtained in several different laboratories, it is difficult to predict with precision the level of expression of the CA150 gene in each of these human tissues from the number of ESTs. However, the fact that human brain cDNA clones make up approximately 11% (8/71 clones) of the human cDNA clones corresponding to CA150 suggests that CA150 is not a high level ubiquitous transcription factor. in all tissues (such as TATA-binding protein, for example), and suggests that CA150 may exhibit preferential activity in certain neuronal subpopulations of the brain. The interest of CA150 is supported by the fact that other PPHs such as HYPM or HYPE (Faber et al., 1998) do not do not seem to be expressed in the human brain due to the absence of ESTs in the human brain corresponding to their genes (Unigene database). The interest of CA150 is also supported by the fact that the htt presents a high proportion of EST in the brain, which reflects an expression of the htt in all the cells of the brain including those which are not reached in HD, while that CA150 has a low proportion of brain EST which is likely to reflect selective expression of CA150 in certain neurons in the brain, possibly those affected in HD.

The inventors therefore set out to highlight molecules capable of mimicking the activity of normal htt or CA150 so as to then use them as a palliative for one or the other of these compounds in order to restore functionality. of the normal htt / CA150 pair. The molecules in question can be any type of compound (natural molecules, synthetic peptides, organic compounds, combinatorial products) capable of mimicking either the activity of normal htt or any N-terminal fragment of normal htt having an activity similar to that of normal htt not only in its interaction with CA150 but also in the consequences of this interaction, in particular with regard to the expressions or repressions of genes induced by the normal functioning of this couple. Preferably, the N-terminal fragment in question will have at most 546 amino acids.

As regards the molecules capable of mimicking the activity of CA150, in the context of the present invention, these molecules also include molecules capable of mimicking the activity of a functional fragment of CA150, that is to say of a fragment capable of fulfilling a function similar to that of CA150 not only in its interaction with normal htt but also in the consequences of this interaction, in particular with regard to the expressions or repressions of genes induced by the normal functioning of this couple. Indeed, the simple overexpression of a functional fragment of CA150, for example a fragment devoid of the region (QA) 38, is capable of allowing the normal functioning of this couple to be restored. Thus, the subject of the present invention is a method for screening molecules capable of modifying the normal htt / CA 150 interaction comprising: a) pre-incubation of the normal htt or of one of its N-terminal fragments with a molecule to be tested, b) the elimination of the molecules to be tested which remained free in the pre-incubation medium, c) the addition to the resulting medium of b) of CA 150 or of a functional fragment of CA150, d) elimination of CA 150 or functional fragment of CA 150 after incubation of the medium c), e) the measurement of the affinity constant of the normal htt couple or N-terminal fragment thereof / CA 150 or functional fragment of CA 150 derived from d), f) the comparison of the affinity constant measured in e) with a standard affinity constant of the normal htt / CA 150 couple.

In order to complete this process or depending on whether the molecule to be tested is suspected of mimicking the normal htt or an N-terminal fragment thereof or CA150 or a functional fragment of CA150, it may be advantageous to "reverse" the reagents used in this process by carrying out the preincubation in a) with CA150 or a functional fragment of CA150 and by adding in c) the normal htt or an N-terminal fragment thereof. Obviously, in this case, it is the normal htt or the N-terminal fragment thereof which remains free in the medium of c) which will then be eliminated in step d). In an advantageous embodiment of the process according to the invention, the compound fixed in step a) of the above process is fixed on solid phase which can be chosen from beads, a tube, a plate, etc.

In fact, the process according to the invention as written above makes it possible to demonstrate a competition occurring between the molecule to be tested and normal htt or an N-terminal fragment of normal htt or else CA150 or a fragment of CA150, depending on the order of the reagents used. Indeed, if the comparison of the affinity constant measured in e) (of the above process) with a finding of standard affinity of the normal htt / CA150 couple reveals an interaction for said couple weaker than that known under standard conditions, then it will be necessary to be interested in the molecule tested which will probably be at the origin of this “loss of interaction” as a palliative with one or the other of the elements of the couple in question. Thus, in order to deepen such data, the method according to the invention may further comprise a step g) in which the affinity constant of the pair tested molecule / normal htt or N-terminal fragment thereof is measured. , or of the couple tested molecule / CA 150 or functional fragment of CA150, and optionally a step h) in which the affinity constant obtained in g) is compared with the standard affinity constant htt normal / CA 150. Such a This approach will indeed make it possible to specify the strength of the interaction of the molecule to be tested with htt or CA150 (or one of their fragment).

In the event that the two above-mentioned steps confirm the potential of a tested molecule to interact at the level of the normal htt / CA150 pair, it is necessary to add to the process according to the invention a step i) in which the tested molecule is identified for which the affinity constant measured in step g) is significant of an interaction with normal htt or an N-terminal fragment thereof or with CA150 or a functional fragment of CA150. This molecule is in fact of particular interest and the present invention also relates to its use for the preparation of a medicament intended for the curative or preventive treatment of Huntington's disease. Indeed, this molecule is capable of restoring in whole or in part a normal functionality of the normal htt / CA150 couple. and thus prevent neuronal apoptosis or neuronal dysfunction induced by mutated htt in the cells concerned. It is not excluded that such a molecule present with normal htt or an N-terminal fragment thereof or with CA150 or a functional fragment of CA150, an affinity constant greater than the known standard affinity constant for the normal htt / CA150 pair. In this case, this type of molecule should be used on purpose.

The present invention also relates to a method of identifying therapeutic targets for Huntington's disease by the comparative analysis of the expression profiles of mRNAs from healthy cells with expression profiles of mRNAs from cells in which Losses of normal htt / CA150 interactions are suspected. By mRNA is meant not only total mRNA but also certain mRNA (by Northern blot, by sequencing of EST from cDNA libraries, by cDNA display, by subtrartive hybridization of mRNA or cDNA, using DNA membranes or microarrays, or any combination of these methods). In the context of the present invention, these mRNAs can be not only derived from cells but also from whole tissues or organisms (human or non-human). When these cells, tissues or organisms are said to be “healthy”, it should be understood: “in the normal state”, that is to say showing no expression of the mutated htt. When it comes to cells, tissues or organisms in which losses of normal htt / CA150 interaction are suspected, it should be understood that this is a pathological condition with expression of the mutated htt. Such a method makes it possible to demonstrate under-expression or over-expression of genes in cells suspected of having a loss of normal htt / CA150 interaction with respect to the expression of the same genes in healthy cells, and the nucleotide sequences corresponding to all or part of these genes can be used in the context of the present invention for the preparation of a medicament intended for the curative or preventive treatment of Huntington's disease.

By “nucleotide sequence corresponding to a part of the gene” is meant any sequence corresponding to a functional fragment of the gene in question and the expression of which is capable of giving rise to a polypeptide having an activity similar to that of said gene at least as regards its action on the functionality of the normal htt / CA150 pair.

By “under-” or “over-expression” is meant any expression of a gene which is lower or greater than normal respectively, which can be detected by standard techniques well known to those skilled in the art.

In the case where the above process reveals that a gene is under-expressed in diseases suspected of having losses of normal htt / CA150 interaction, the above use will consist in introducing the nucleotide sequence corresponding to all or part of this gene in the cells concerned in a construction containing all the elements necessary for the expression of a gene, so as to re-express this gene in situ and thus restore the functionality of the normal htt / CA150 pair. Such an approach can for example be carried out by gene therapy. In the case where the method described above makes it possible to demonstrate an overexpression of a gene in cells suspected of having losses of normal htt / CA150 interaction, the above use will consist in blocking the expression in situ of this gene for example by means of an antisense strategy. The present invention also relates to the use of a nucleotide sequence corresponding to the CA150 gene or of a functional fragment of CA150 in order to overexpress it by means of a construction comprising all the elements necessary for this overexpression, level of cells suspected of having normal htt / CA150 interaction losses, as part of a curative or preventive treatment for Huntington's disease. Indeed, in the case where normal CA150 is deficient or in the case where pathological cells contain both mutated htt and normal htt, such an approach consisting in locally over-expressing CA150 would have the effect of shifting the balance of interactions by saturation of the mutated htt and would therefore make available the C Al 50 in excess for the normal htt present in said cells.

This could also be the case with hyperactive analogs of CA150 capable of restoring functionality at least equivalent to the standard functionality of the normal htt / CA150 pair. The present invention also relates to an anti-mutated htt antibody capable of discriminating between normal htt and mutated htt. It also relates to the use of such an antibody for the preparation of a medicament intended for the curative or preventive treatment of Huntington's disease. Such a process of “neutralization” of the mutated huntingtin could moreover be entirely accompanied by a local over-expression of CA150 or a functional fragment of CA150 and / or of normal htt or of an N-terminal fragment. from normal htt so as to reintroduce the partners necessary for restoring the functionality of the normal htt / CA150 pair. As an example, this could be achieved through gene therapy.

The present invention is not limited to the above description. It will be better understood in the light of the experiments set out below.

PRESENTATION OF THE FIGURES

Figure 1. Quantitative test (ONPG test in liquid phase) for the interactions between N-terminal fragments of the htt and the protein ZK1127.9 187-758. Htt90 corresponds to the product of exon 1 of the htt gene. Htt 152 is a fragment that is more likely to enter the neuron nucleus than htt546. The values represented correspond to the means over 45 in vivo measurements (9 independent cultures; 5 measurements / point / culture). The intervals represent the limits of the values obtained. Δ: percentage of link difference between the 15 Gins allele and the 128 Gins allele. The distributions of ONPG values were compared by ANOVA analysis using Macintosh Statview software.

Figure 2. Comparison of the proteins ZK.1127.9 and CA150. The annotated sizes are in number of amino acids. Black squares: WW domains. Hatched area: polymorphic domain (QA) 60 in the normal population, 4 alleles, at least 15% - 30% heterozygosity, depending on the normal population tested. Figure 3. In vitro interaction of normal htt 152 (fixed on sepharose beads in the form of GST fusion protein) and ZK1127.09_187- 758:: HA. The interaction is demonstrated using an anti-HA antibody. -httl52Q18: no normal htt on the sepharose beads (negative control); + httl52Q18: with normal htt on the sepharose beads, in this case the arrow indicates the protein ZKl 127.9 eluted after retention by the htt fixed on the beads.

Figure 4. In vitro interaction of GST:: normal htt152 (fixed on sepharose beads in the form of GST fusion protein) and CA150 (fragment 133-910 amino acids):: HA. The interaction is demonstrated using an anti-HA antibody.

Figure 5. Delay in migration on CA150 gel (fragment 133-910 amino acids) after expression and purification in E. coli. CA150 is detected using an anti-HA antibody or a polyclonal anti-CA150 antibody. A: detection with an anti-HA antibody (commercial origin). B: detection with a polyclonal anti-CA150 antibody (Sune et al., 1997). Ag: aggregated form; N.Ag: non-aggregated form.

Figure 6. Expression profile of the CA150 protein on post-mortem brain sections of normal individuals and patients with Huntington's disease. The expression of CA150 was tested using a polyclonal anti-CA150 antibody (Sune et al., 1997). The tests were carried out in Robert Ferrante's laboratory (Bedford VA medical Center, Bedford, USA). CA150 has the following expression profiles: A: Human cortex, normal individual: diffuse expression, mainly nuclear. B: Human cortex, patient with Huntington's disease: expression in the form of aggregates in the nucleus and in the cytoplasm of positive neurons. This expression profile is strongly similar to that observed with an anti-huntingtin antibody (as widely described in the literature), which suggests that CA150 and huntingtin co-localize in cellular aggregates in patients. C: human striatum, normal individual: same observation as for A. D: human striatum, patient suffering from Huntington: same observation as for B. When the polyclonal anti-CA150 antibody is pre-incubated overnight with the purified CA150 protein (preabsorption test), no signal is visible, which demonstrates the specificity of the markings observed in A, B, C and D.

METHODS AND DATA

The constructs intended for double-hybrid screening and encoding the htt were created in the vector pGBT9 (Genbank accession number UO7646, Clontech) in the form of fusion proteins with the binding domain of the GAL4 protein (GAL4BD) for the specific DNA sequences that have been introduced into the genome of host yeasts. The cDNA fragments coding for htt are from the laboratory of Michael Hayden, University of British Columbia, Vancouver, Canada. All the constructions were sequenced in order to verify the reading phase of the cDNAs of the htt, the number of CAG repeats, and the absence of point mutations or deletions. The C. elegans cDNA library (cDNAs generated using an oligo-dT primer) was subcloned into the vector pACT2 (Genbank accession number U29899, Clontech) in the form of fusion proteins with the GAL4 transcription activation domain (GAL4AD). This cDNA bank is freely accessible and comes from the laboratory of R. Barstead (Oklahoma Medical Research Foundation, OK, USA). The double-hybrid screening was carried out using a protocol based on the fusion of mat-a and mat-alpha yeasts (Bendixen et al., 1994), the mat-a yeasts (strain CGI 945, Clontech) expressing in this case the fusion proteins [GAL4BD :: htt], and the mat-alpha yeasts (strain Y187, Clontech) expressing the fusions [protein fragments of

The construct used by the inventors for screening (fusion protein with GAL4BD) was htt546Q15 (Figure 1). After screening 3.8 10 ⁸ clones (fusion efficiency greater than 33%), 56 positive clones were detected. After elimination of false positives (based on a double-hybrid interaction with the fusion protein GAL4BD :: lamine) and verification of the redundancy of the true positives by analysis of the restriction profiles (enzymatic digestion of the cDNAs selected using the restriction enzymes EcoRI and BamHI), the inventors sequenced the 5 'and 3' ends of the unique cDNA clones results (i.e. 2 clones). Search for sequence homology between the 3 'and 5' ESTs (Expressed Sequenced Tag) obtained for these clones and the proteins of C. elegans on the "BLAST C. elegans" server at the Sanger Center, Cambridge, UK (http: //www.sanger.ac.uk/Projects/C_elegans/blast_server.shtml) using BLAST programs (Altschul et al., 1997) enabled the inventors to identify the protein ZKl 127.9 as a partner of htt546Q15. As indicated in the Acedb public database, the gene for ZKl 127.9 is found on chromosome II (cosmid ZKl 127) of C. elegans. The sequence of the protein ZKl 127.9 was predicted from the analysis of the genomic sequence of C. elegans (The C. elegans Sequencing consortium, 1998) and contains 946 amino acids. Comparison of the 3 'and 5' end sequences of the cDNA clone corresponding to ZKl 127.9 with the C. elegans library of the Sanger Center using the BLASTN and BLASTX programs made it possible to establish that the cDNA clone corresponding to the gene for the protein ZKl 127.9 codes for amino acids 187 to 758 of this protein. The binding sites of htt546Q15 on ZKl 127.9 are therefore located between amino acids 187 to 758 of ZKl 127.9 (ZKl 127.9_187-758). The interaction profile of ZKl 127.9 187-758 with different forms of htt (htt546Q15, htt546Q128, httl52Q15, httl52Q128) was analyzed by the inventors using double-hybrid tests (Bendixen et al., 1996) , initially qualitatively after selection of positive clones on agar and transfer to nylon filters, and then quantitatively after selection of positive clones in liquid phase, protein extraction, and ONPG test in liquid phase (Kalchman et al., 1996 ). The ONPG tests were carried out 45 times, ie from 9 independent yeast cultures, and from 5 measurements / case / culture (Figure 1). Statistical analysis of the data was performed using Statview software (Macintosh) and analysis of variances on the distribution of values obtained for each case (Figure 1). Sequences of ZKl 127.9 and ZKl 127.9_187-758 with other genes have been sought by the inventors in several public databases, including:

- Genbank at NUL MD (http://www.ncbi.nlm.nih.gov) - WWW server "BLAST C. elegans" from the Sanger Center, Cambridge, UK (http://www.sanger.ac.uk/ Projects / C_elegans / blast_server.shtml) the EBI WWW server, Cambridge, UK (http://www2.ebi.ac.uk/servicestmp)

This research was carried out using BLAST programs (Altschul et al, 1997) or using programs based on the Smith and Waterman algorithm (Smith and Waterman, 1981) such as the bic2 program (EBI). In addition, conserved domains were searched in ZKl 127.9, ZKl 127.9 87-758, and CA150 using the Pfam program (http://www.sanger.ac.uk/Softwares/Pfam search.shtml). These analyzes made it possible to highlight the following facts (Figure 2):

- ZKl 127.9 187-758 contains 3 WW domains (Pirozzi et al., 1997) at positions 200-225, 262-292, and 378-407 amino acids (0.00019 <Evaluated <0.011). The WW domains are 35-40 amino acid motifs characterized by the presence of 4 conserved aromatic residues, two of which are tryptophans, mediate protein-protein interactions, and conventionally interact with polyproline domains (Pirozzi et al, 1997 ). It is therefore very likely that ZKl 127.9_187-758 interacts with the proline-rich region of the htt (htt: Genbank accession number L 12392) which is located immediately at the C-terminal of the polyQ domain (between amino acids 40 and 100) and which contains 2 polyprolines of 11 and 10 consecutive prolines. It is also very likely that the binding of htt to CA150 involves the polyprolines and the immediately C-terminal region (on at least 30 amino acids) to the polyproline domains in the htt and the WW domains at positions 133-162, 431-460 ( corresponding to the domain WW 262-292 of ZKl 127.9), and 532-559 aminocides (lO ^ EvaluesO.0016) of CA150. The mediating role of the polyproline-rich region of htt for interaction with WW domain proteins has been illustrated elsewhere (Faber et al, 1998). - the region of ZKl 127.9 going from amino acids 267 to 944 has a significant homology (identical amino acids: approximately 38%; similar amino acids: approximately 69%; p = 1213 in Smith and Waterman analysis, and p (N) = 10 ^{" 137} in BLAST analysis) with the region 435 to 1095 amino acids of the human protein CA150 (Genbank accession number AFO 17789). There are also other weaker and non-significant homologies for short segments of ZKl 127.9, such as for example homologies with WW domains present in other proteins such as HYPA or HYPB (Faber et al, 1998).

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Claims

1. A method for screening molecules capable of modifying the normal htt / CA150 interaction comprising: a) pre-incubation of the normal htt or one of its N-terminal fragments with a molecule to be tested, b) elimination molecules to be tested remained free in the pre-incubation medium, c) the addition to the resulting medium of b) of CA150 or of a functional fragment of CA150, d) elimination of CA150 or functional fragment of CA150 after incubation from the middle c), e) the measurement of the affinity constant of the normal htt couple or N-terminal fragment thereof / CA150 or functional fragment of CA150 from d), f) comparison of the affinity constant measured in e) with a standard affinity constant of the normal htt / CA150 couple.

2. Method according to claim 1 in which: the pre-incubation in a) is carried out with C Al 50 or a functional fragment of CA150, in c) the normal htt or N-terminal fragment thereof is added, and in d) the normal htt or N-terminal fragment thereof which remains free in the medium of c) is eliminated.

3. Method according to claim 1 or 2 in which step a) is carried out by fixing the normal htt, an N-terminal fragment thereof, CA150 or a functional fragment of CA150, on solid phase

4. Method according to one of claims 1 to 3, further comprising a step g) in which the affinity constant of the pair tested molecule / normal htt or N-terminal fragment thereof or CA150 or functional fragment is measured of CA150, and optionally a step h) in which the affinity constant obtained in g) is compared with the standard affinity constant htt normal / CA150.

5. Method according to claim 4 further comprising a step i) in which the tested molecule is identified for which the affinity constant measured in step g) is significant of an interaction with normal htt or N-terminal fragment of this or CA150 or functional fragment of CA150.

6. Use of the molecule identified according to claim 5 for the preparation of a medicament intended for the curative or preventive treatment of Huntington's disease.

7. Method for identifying therapeutic targets for Huntington's disease by comparative analysis of expression profiles of mRNAs from healthy cells with expression profiles of mRNAs from cells in which normal htt interaction losses / CA150 are suspected.

8. Use of a nucleotide sequence corresponding in whole or in part to a gene under-expressed in cells suspected of having losses of normal htt / CA150 interaction with respect to healthy cells in accordance with the method according to claim 7, for the preparation of a medicament intended for the curative or preventive treatment of Huntington's disease.

9. Use of a nucleotide sequence corresponding in whole or in part to a gene overexpressed in cells suspected of having losses of normal htt / CA150 interaction with respect to healthy cells according to the method according to claim 7, for the preparation of a medicine intended for the curative or preventive treatment of Huntington's disease.

10. Use of a nucleotide sequence corresponding to the CA150 gene or of a functional fragment of CA150 for the preparation of a medicament intended for the curative or preventive treatment of Huntington's disease by overexpression of said sequence in suspected cells to show normal htt / CA 150 interaction losses.

11. Anticoφs anti-htt mutated.

12. Use of an anticoφs according to claim 11 for the preparation of a medicament intended for the curative or preventive treatment of Huntington's disease.