FR2808968A1 - New transgenic nematode with dysfunctional neuronal phenotype, useful for identifying therapeutic targets associated with polyglutamine expansion diseases - Google Patents

Abstract

A transgenic nematode (A) having a dysfunctional neuronal phenotype without neuronal death at the young adult stage, is new. Independent claims are also included for the following: (1) transgene (I) comprising a promoter specific for a touch-sensitive neuron linked to a gene encoding a protein (II) that includes a polyglutamine (pQ) domain; and (2) screening for therapeutic targets for treatment of pQ-expansion disorders.

Description

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 The present invention relates to a transgenic nematode having a phenotype of neuronal dysfunction in the apparent absence of neuronal death. The invention also relates to the transgene at the origin of said phenotype as well as methods of screening therapeutic targets for the treatment of polyglutamine-expanding diseases.

 The first characteristic of abnormally sized polyglutamines (or polyglutamines) is their pathological effect when they are present in certain proteins, which today determines a class of eight autosomal dominant neurodegenerative diseases. The number of units of glutamine (or gln) and the protein context are two elements which intervene in the pathological effect of polygln sequences.

 These eight polygln expanding neurodegenerative diseases are: Huntington's disease (HD, The Huntington's Disease Collaborative Research Group, 1993), spinocerebellar ataxia (SCA) type 1 (Orr et al., 1993), SCA2 (Imbert and al., 1996), SCA3 or MachadoJoseph's disease (Kawagushi et al., 1994), SCA6 (Ikeuchi et al., 1997), SCA7 (David et al., 1997), Atrophy Dentatro-Rubro Pallidoluysienne DRPLA (Koïde et al., 1994) and Spino-Bulbar Muscle Atrophy SBMA (La Spada et al., 1994). The SCA6 gene has the particularity of presenting a moderate pathological threshold compared to other diseases, probably due to the position of the polygln (transmembrane) in the calcium channel [alpha] 1A. The functions of disease proteins are only known for two of these diseases: ion channel for SCA6, and androgen receptor for SBMA. These neuropathies are rare and incurable. Such diseases greatly disturb the social and family life of the patient because they often appear.

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 at the age of 30-40 years. They are very physically debilitating but also difficult to manage by loved ones because the patient has serious psychological and psychiatric disorders. The disease progresses over a period of 15 to 20 years.

 For example, the prevalence of HD in the French population is around 1 / 10,000. The HD protein is huntingtin (htt), its expression is ubiquitous, its localization is apparently cytoplasmic, and its molecular weight is 350 kDa. The htt is characterized by the presence in its N-terminal part (N-ter) of a polygln sequence followed by two poorly polymorphic polyproline regions.

The normal htt contains a normal number of glutamine repeats (typically: 15 to 20 gln, mainly less than 39 gln) The mutated htt contains an abnormally high number of glutamine repeats (typically the pathological threshold is around 39 gin, the expansion polygln, for example up to 180 gln in some cases) which is associated with the onset of the disease (Koshy and Zoghbi, 1997). The biochemical and cellular function of htt is still unknown. Immunohistochemistry, electron microscopy and cell fractionation techniques have made it possible to observe that htt is a cytosolic protein associated with vesicles and / or microtubules, which suggests a role in anchoring to the cytoskeleton or in the transport of vesicles (Sathasivam et al., 1999). Studies using homozygous / / for HD mice (the murine counterpart of the human gene IT15) have made it possible to suppose that the htt has an important role during embryogenesis because the death of the embryo occurs after only a few days in these mice (Zeitlin et al, 1995). Very recently, a team working on embryonic cells deleted from HD mice proposed that htt could have an important role during hematopoiesis (Metzler et al., 2000). These three observations allow us to sketch a diagram of the functioning of htt where this protein is important in several stages of development.

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In general, the neurodegeneration profiles of the brain are specific for the disease while the expression of the disease proteins is ubiquitous. The mechanism of this neuronal selectivity is still not elucidated (Koshy et al., 1997). Polygln-expanding neurodegenerative diseases have in common the presence of intranuclear (NII) or even cytoplasmic inclusions. These inclusions have been demonstrated by immunohistochemistry on post-mortem brain sections for HD (DiFiglia et al., 1997), SCA1 (Skinner et al., 1997), SCA3 (Paulson et al., 1997), SCA6 ( Ishikawa et al., 1999), SCA7 (Holmberg et al., 1998), DRPLA (Igarashi et al., 1998) and SBMA (Li et al., 1998a). These studies have shown that the inclusions consist of fragments of disease proteins but also of ubiquitin. The role of inclusions, especially those that are nuclear, as a cause in these diseases remains very uncertain.

 The polygln sequences also have self-aggregating properties which have been described by Perutz et al. in 1994. In this model, the polygln behave like polar zippers, where the side chains of the gln form P sheets via hydrogen bonds, the stacking of sheets (3 of several proteins leading to a phenomenon of These aggregates seem to be irreversible.

The observation of a significant migration delay on polyacrylamide gel of fusion proteins [GST-N-ter fragment of htt with 30.51 or 83 gln] shows the ability of polygln proteins to self- aggregate and the great difficulty in dissociating them by heating and using the SDS (Wanker et al., 1999; Hollenbach et al., 1999). It would seem that the formation of aggregates, at least in vitro, follows a kinetics which is a function of the size of the polygln and its concentration (Scherzinger et al., 1999) In addition, the size of the protein carrying the polygln also influences the formation of aggregates because the larger the protein, the higher the polygln

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 seems to be stabilized in a state where its capacity to form aggregates is minimal Indeed, the expression of the mutated htt, whole or truncated, in a line of neuroblastoma showed in the first case a diffuse and cytoplasmic expression and, in the second case, cytoplasmic and nuclear aggregates (Cooper et al., 1998).

 So not only does the presence of a polygln of a certain size in a disease protein generate the formation of aggregates which modifies the conformation of the protein, but it is likely that this results in the modification of interactions with d other proteins. Thus, the htt undergoes an increased cleavage by caspase-3 when the protein is mutated.

This proteolysis releases an apparently very toxic N-ter fragment containing the polygln and the two polyproline domains (Goldberg et al., 1996).

 The aggregates observed in HD consist of Nterminal fragments of htt, ubiquitin, and other proteins. Several proteins are likely to be sequestered in aggregates if they interact in one way or another with the N-terminal part of htt: for example, the SH3GL3 protein with SH3 domain interacts better with htt mutated by l 'intermediate polyproline domains and it is associated with aggregates (Sittler et al., 1998). Transglutaminases, enzymes which participate in post-transcriptional modifications, could also intervene in the formation of aggregates A network of proteins would be formed from polygln proteins entangled with each other by covalent bonds between glutamines and lysines In the presence of transglutaminases , a synthetic peptide of 18 polygln shows the capacity to form aggregates in vitro (Kahlem et al., 1996). Nevertheless, it is likely that the ability of polygln to form polar zippers is more strongly determining in the formation of aggregates. It is interesting to note that the co-aggregation of the mutated htt with other polygln proteins has been demonstrated. , for example in COS cells with the

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 CBP protein (CREB-Binding-Protein) which contains 19 gln (Kazantsev et al., 1999).

Aggregates in HD are present at different levels in neurons: intranuclear inclusions (NII), cytoplasmic aggregates and aggregates at the axon ends (Ross et al., 1999). The latter type of aggregate is somewhat special since it is smaller than the others and does not contain ubiquitin (Li et al., 1999). In addition, there is a better correlation between the development of neurodegenerative symptoms and axonic aggregates in transgenic mice than with NIIs. They are detected earlier and are more numerous than NIIs, and their number continues to increase with the advancement of the pathology. Axonic aggregates could be responsible for deficits in neuronal transmission and communication (Li et al., 1999). After focusing on the central nervous system, the search for aggregates was extended to other tissues. Using the antibody CAG53b directed against the N-terminal part of the htt and an antibody directed against ubiquitin, aggregates were detected in the muscle fibers, the heart muscle, the hepatocyte cells, and the Langerhans cells of the murine transgenic line R6 / 2 expressing exon 1 of the IT15 gene. In addition, the presence of aggregates in the organs correlates with the decrease in their size (Sathasivam et al., 1999). In this transgenic model, the mice are diabetic with a blood sugar level twice higher than normal (Hurlbert et al., 1999). The fact that these mice are diabetic because of the mutated htt is however not clearly established
The mutated htt leads to cellular dysfunctions which lead to neuronal death, the profile of which resembles neither that of necrosis nor that corresponding to programmed cell death (Mangiarini et al., 1996). Disturbances in neuronal communication (decrease in the production of neurotransmitters or their receptors) are likely to be induced in neurons affected before

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 cell death. The chronology of events observed in mouse models and invertebrate model organisms will allow a better understanding of the pathological process involved in HD. The best characterized mouse model is the transgenic line R6 / 2, which expresses exon 1 of the IT15 gene with 144 gln (Mangiarini et al., 1996). However, as noted above, these mice are sick with diabetes. The stages of the pathological process in the R6 / 2 model follow one another as follows: detection of neuronal NIIs at 4 weeks (Davies et al., 1997), onset of the first symptoms between 9 and 11 weeks (ex: dyskinesia associated with onset loss of brain and body weight), then rapid death of the animal between 10 and 13 weeks (Mangiarini et al., 1996). More recently, two transgenic mouse models expressing the whole mutated htt have been described (Reddy et al., 1999; Hodgson et al., 1999). These models are probably more representative of HD than the R6 / 2 model insofar as the whole htt was used, and insofar as the mice are not sick with diabetes. The mutated polygln sizes are roughly the same in the two models (around 45 gln and 80 gln). Although the neurological phenotypes appear around 28 weeks, the presence of NIIs does not correlate in the two models with the neurological phenotype induced that of Reddy et al. allows to observe NIIs at 12 weeks, whereas that of Hodgson et al. does not demonstrate the presence of NII in the affected neurons.

 Neural aggregates are in fact common to several neurodegenerative diseases including diseases other than those associated with polygln such as prion diseases or Alzheimer's disease. In the group of polygln diseases, the only factors of variation are the nature of the polygln carrier protein and the location of the sites of neuronal loss. The study of pathological mechanisms using animal models suggests that aggregates are an initiator of the disease process because they appear before the onset of symptoms. In

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 In humans, the presence of aggregates seems to correlate with the severity of HD since the study of post-mortem sections shows that the number of NIIs is a function of the size of the polygln (Aronin et al., 1999). However, a recent study disputes the correlation between distribution of aggregates and severity of the pathology, because the use of immunohistochemistry techniques on post-mortem brain sections of patients with HD showed a distribution of aggregates which did not correspond to the severity of neurodegeneration. The primary area of degeneration in HD is the striatum and then the cortex, while the distribution of aggregates is greater in the cortex (Kuemmerle et al., 1999). The aggregates can have a toxic role in the pathological process by sequestering proteins having an important function for the cell.

 A loss of function due to the sequestration of proteins essential for neuronal survival is another hypothesis which would imply aggregates in a more indirect and more properly fortuitous way. Similarly, the sequestration of a toxic form of the protein disease (protective mechanism) such as the Nterminal fragment of the mutated htt is also likely. The presence of polygln causes a conformational modification of the disease protein giving it new interaction properties, in particular with caspase-3 and releases an N-terminal fragment (Goldberg et al., 1996). The normal htt is localized in the cytoplasm in free form while the Nterminal fragment is localized in the nucleus in the form of NII. Due to its small size (less than 50-70 kDa), this truncated and mutated protein could passively diffuse into the nucleus and interact abnormally with nuclear proteins, RNA and DNA. These new properties would give it a potentially toxic role. The formation of aggregates mainly by this N-terminal fragment of the htt would limit its toxicity. This hypothesis is supported by the inhibition of caspase-1,

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 acting upstream of caspase-3, and which in R6 / 2 prolongs the life of the mouse and delays the appearance of bodies of inclusions as well as alterations of neurotransmitter receptors and the onset of symptoms (Ona et al., 1999). Likewise, the use of anti-apoptotic compounds or neurotrophic factors in the lines of striatal neurons and the blocking of the nuclear localization of the N-terminal fragment protect from cell death and reduce the number of aggregates (Saudou et al. , 1998).

 It therefore appears that NIIs in HD can be considered toxic, limiting, or even protective of cell death. It is very important to note that while the cause and effect relationship between NII (a seemingly late form of aggregation) and neuronal death has been widely studied, it remains subject to several criticisms. There is limited data on causal relationships between cytoplasmic aggregates in the cell body (which are thought to be earlier) and those in axons (which are more toxic than smaller), neuronal death and, more interestingly, with dysfunction except apoptosis. The pathological mechanisms of HD and of all polygln neurodegenerative diseases are therefore not elucidated, and current knowledge does not allow us to favor one or the other of the hypotheses according to which the aggregates (in the broad sense and whatever or their location) cause or protect against neuronal deficits.

 There is therefore a real need for reliable animal models not only for the study of the mechanisms of initiation and development of neurodegenerative diseases but also for the determination of compounds capable of being used for treatment and / or prevention of these diseases.

 Work has thus been carried out in mice. Indeed, several studies of neurodegenerative disease proteins in mice have made it possible to observe the induction of a neuronal phenotype by abnormally sized polyglutamines, for example expression in mice

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 transgenics of exon 1 of the IT15 gene mutated with approximately 144 gln (Mangiarini et al., 1996). In all of these studies, it is observed that the severity of the phenotypes is a function of the size of the polyglutamine sequence. In order to test the intrinsic properties of the mutated polygln, a study in mice also consisted in introducing a mutated polygln in a foreign context, the protein hypoxanthine phosphoribosyltransferase (HPRT: enzyme involved in the metabolism of purine). A neurodegenerative phenotype was observed for 146 gln but not for 70 gln (Ordway et al., 1997). However, the mouse models do not make it possible to search, by rapid genetic screening and without a priori on the genes involved, genetic modifiers of the effects of polyglutamines.

 As such, the use of simple model organisms (WHO) has many advantages. Indeed, these models are very easy to handle (superior to the mouse) because of their size and because of the duration of their reproductive cycle. These are, for example, nematodes or Drosophila flies. In addition, the Caenorhabditis elegans and Drosophila melanogaster models have a good degree of genetic conservation of several important cellular mechanisms in humans. They have a rudimentary nervous system for C. elegans and more advanced for D. melanogaster, allowing to study neurodegenerative diseases.

 But 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, numerous 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,

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short reproductive cycle (a few days), knowledge of complete cell phylogeny, ease of use (culture without solid or liquid agar, resistance of larvae to freezing, and possibility of high-throughput screening, for example in 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, the study of the mechanisms of human diseases in C. elegans is recognized as a tool for powerful study for the identification and validation (at the cellular level, and before anatomo-functional pharmacological study in more complex models such as mice or primates) of therapeutic targets.

 These animal models are used in the field of the invention from a therapeutic perspective because their use lends itself to "rapid" research of suppressor mechanisms. After having modeled a disease in a WHO, the screening of a sick population subjected to a mutagen can be undertaken in order to select the individuals presenting a reversion of the sick phenotype. WHO, such as the nematode or Drosophila, is devoid of most polygln disease proteins, and the search for suppressor mechanisms by the use of transgenic WHO expressing genes for human diseases is innovative (Jackson et al., 1998, Faber et al., 1999; Marsh et al., 2000).

 Each of these models, however, has its limits and / or its specificities. Indeed, in Drosophila, the phenotypes induced by the mutated polyglutamines are of the neuronal dead type (neurodegeneration) and in the nematode C. elegans, the phenotypes induced by the mutated polyglutamines have so far been weakly penetrating which did not allow the search for suppressive mutations in effectors of these phenotypes.

 However, based on the highly penetrating phenotypes of some suppressive mutations, the effects of the mutated polygln on death

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 neuronal have been recently identified in Drosophila Thus, the coexpression of the candidate gene p35, caspase inhibitor, has shown a beneficial effect on neuronal degeneration in foreign flies for "SCA3" (Bonini 1999), but does not lead to a decrease in the neurodegenerative phenotype in transgenic flies for "HD" (Jackson et al., 1998). The signaling pathways involved may not be the same in SCA3 and HD. Another study concerns the Hsp70 (Heat shock protein) chaperone of D. melanogaster which has been detected in nuclear inclusions in "SCA3" foreign flies. The co-expression of the human counterpart of Hsp70 (HSPA1L) and of ataxine-3 (protein of the disease SCA3) containing 78 gln also made it possible to inhibit the phenotype of neurodegeneration without reducing the number of nuclear inclusions ( Warrick et al., 1999). It may also be that co-expression of a protein with a whole and normal polyglutamine domain is sufficient to counteract the effects of the mutated truncated protein in Drosophila.

 Using C. elegans, a group in the United States was able to demonstrate poorly penetrating phenotypes induced by the mutated polyglutamines in transgenic animals (WO 99/02652 and Faber et al., 1999). This model does not allow the search for new therapeutic targets by reversing the transgenic phenotypes induced by the mutated polyglutamines due to their low penetrance.

 The inventors therefore set out to highlight a phenotype of neuronal dysfunction in lines of C. genecans transgenic nematodes which is representative of the supposedly early stages of neurodegenerative diseases (neuronal dysfunction before neurodegeneration) and which has the advantage of having a strong penetrance.

 The present invention therefore relates to a transgenic nematode having a phenotype of neuronal dysfunction in the absence

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 apparent neuronal death at least in the young adult stage, which corresponds to worms 3 to 4 days old. This phenotype has the advantage of having, in particular at this stage, a penetration of at least 50% or even at least 70%. By penetrance is meant the frequency with which the gene manifests its effects.

 More particularly, the transgenic nematode according to the invention comprises a construction allowing the expression of a protein having a polyglutamine domain under the control of a promoter specific for neurons. According to an advantageous embodiment, the above promoter is specific for touch receptor neurons. The inventors have in fact carried out mechanosensory analyzes on transgenic nematodes expressing polyglutamine expansions of different lengths. To do this, they used the promoter from the mec-3 (mec-3p) gene (Chalfie and Au, 1989) to direct the expression of the transgene in 10 of the 302 worm neurons, including the 6 receptor neurons. sensation (or touch): AVM, ALML, ALMR, PVM, PLML and PLMR. These neurons are the source of responses in worms in the case of a gentle stimulus by touch (Driscoll M., Kaplan J, 1997).

 The inventors' work mainly focused on a polyglutamine domain protein representative of the neurodegenerative diseases that is huntingtin, and in particular on an N-terminal fragment of huntingtin. Thus, in the context of the present invention, by protein with polyglutamine domain, it is understood indifferently means protein with polyglutamine domain or peptide derived from a protein with polyglutamine domain having the expansion of polyglutamine.

 The inventors therefore set out to detect a posterior or anterior mechanosensory phenotype (MEC) in transgenic nematodes in which they induced neuronal dysfunction by causing some of these animals to express, at the level of target neurons,

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 of a mutated polyglutamine domain protein. This phenotype is expressed by the loss of sensitivity to touch due to an alteration of the above neurons. Thus, the inventors sought to detect the transgenic nematodes in which the mutated polyglutamine domain protein was expressed and this expression is detectable by observing the response of the worms or transgenic nematodes to the touch. Indeed, the expression of a polyglutamine domain protein mutated at the level of the aforementioned neurons causes a dysfunction of these neurons. The polyglutamine domain protein implemented by the inventors is huntingtin which, when mutated, contains as previously reported, an abnormally high number of polyglutamine repeats that are typically around at least 39 glutamine residues. In the context of the present invention, the polyglutamine domain comprises from 39 to 128 glutamine residues, preferably between 88 and 128 glutamine residues and more preferably 128 glutamine residues.

 The above-mentioned construction therefore notably includes the nucleic sequence coding for the first 29 amino acids of the mec-3 protein. However, it is possible that this construction works with less than 29 amino acids or even no amino acid of the mec-3 protein.

 In the context of the present invention, the inventors show that the expression of a polyglutamine under the control of the mec-3 promoter at the level of the PLM neurons indicates an insensitivity to touch at the level of the tail of the animals with a penetrance of at least at least 50% or even at least 60% and even at least 70%. In the present case, the tests were carried out on 5 independent stable transgenic lines. The promoter of the mec-3 gene codes for a transcription factor expressed, as indicated above, in 10 target neurons including the 2 PLM neurons which exclusively control the response to touch at the tail. This phenotype is apparently not accompanied by the death of neurons

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 targets (based on observations by NOMARKSI microscopy and on the absence of nuclear labeling with acridine orange), or at the time of measurement of the transgenic phenotype in animals in the young adult stage (3 to 4 days after birth ), or later in the same older animals (7 to 8 days after birth).

 Thus, the inventors have produced a transgene comprising in particular a construct which itself comprises a nucleic sequence coding for the first 29 amino acids of the protein Mec-3, then coding for the first 17 amino acids of huntingtin, then coding for the domain polyglutamine comprising from 39 to 128, preferably from 88 to 128, more preferably 128 glutamine residues, then coding for the 17 amino acids of huntingtin according to the polyglutamine domain, the whole being preceded by the nucleic sequence of the promoter of the gene mec- 3.

By nucleic sequence of a promoter, it is necessary to understand any nucleic sequence corresponding to the entirety of said promoter but also to any functional part of this promoter, that is to say capable of fulfilling a function similar to that of the whole promoter
In addition, in order to allow visualization of the expression of the mutated protein in the transgenic nematode, the inventors have, in a preferred embodiment of the present invention, made sure that said protein is expressed in fusion with a protein. allowing its visualization. Thus, the transgene according to the invention comprises the construction as defined above, the nucleic sequence of which codes, following the protein with a polyglutamine domain, for a protein allowing the visualization of the resulting fusion protein. Advantageously, the protein allowing the expression of said fusion protein to be visualized is the Green Fluorescent Protein (GFP). The present invention therefore also relates to this type of

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 transgene but also transgenic nematodes having been transformed by this type of transgene.

 The processes for transforming nematodes are not described in the context of the present invention because they are now completely within the reach of normal knowledge of a person skilled in the art. Indeed, as indicated above, transgenic nematodes have already been produced and, on this point, the present invention is not distinguished from the prior art. Indeed, it is clear from the present application that the invention resides in particular in the choice of the constituent elements of the construction corresponding to the strangene according to the invention, and in particular, in the promoter chosen by the Inventors.

 At first, the Inventors actually carried out the following construction: mec-3p :: Htt1-57 (84Q) :: GFP. Nematodes transformed with this transgene express, under the control of the promoter of the mec-3 gene, 57 amino acids of huntingtin (Htt) (i.e. the first 17 amino acids, 23 glutamine residues and the 17 amino acids according to the polyglutamine domain) with a polyglutamine expansion of 84 (84Q) fused to the green fluorescent protein (Green Fluorescent Protein or GFP). The animals have a posterior MEC phenotype (insensitivity to touch) of around 35% at the young adult stage (3 to 4 days after birth), which increases to around 50% when the worms are 7 days old, without evidence of death. cellular. From these first results, the inventors sought to develop this system and they showed in particular that the expression under the control of the mec-3 promoter of a polyglutamine of 128 gln in a fusion protein MEC3 :: Htt :: GFP in PLM neurons induces insensitivity to touch in the tail of animals expressing the above protein in 60 to 80% of cases.

 Consequently, the transgenic nematode according to the present invention constitutes an extremely useful animal model for genetic screening and the search for suppressive mutations in genes.

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 effectors, a model which seems more particularly representative of the effects of mutated polyglutamines on neuronal dysfunction before neuronal death. Preferably, the transgenic nematode according to the invention is Caenorhabditis elegans.

The characteristics of this model are interesting for several reasons:
1) the strength of the Mec phenotype makes it possible to search for suppressive mutations in the transgenic lines expressing a large number of gln,
2) several studies strongly suggest that neuronal dysfunction before neuronal apoptosis causes the onset of symptoms in patients,
3) the poor correlation observed by the inventors between on the one hand the formation of nuclear or peri-nuclear aggregates (visible in GFP fluorescence microscopy and in confocal microscopy) and, on the other hand, the anomalies of the Mec phenotype, suggests that 'there is no cause and effect relationship between nuclear or perinuclear aggregates and neuronal dysfunction.

 This new observation complements recent data from the literature where aggregation formation and neuronal apoptosis are not in perfect correlation (Kuemmerle et al., 1999; Reddy et al., 1999). In addition, the inventors have observed that the number of aggregates in the axons of the target neurons (PLM neurons) increases with the size of the polyglutamine, which could suggest a new mechanism (axonal and consequently synaptic deficiencies) for the dysfunction of these neurons in terms of response to the affected tail.

 On the basis of point 1), it may be a question of seeking, after mutagenesis of the transgenic nematodes expressing the mutated htt, a partial or total recovery of the loss of neuronal function induced by

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 the mutated htt (to the F2 or F3 generations). These screenings will be carried out according to conventional techniques (mutagenesis with EMS, majority of nonsense mutations) from a line whose transgene is in the form of extra-chromosomal copies (stable line not integrated). The suppressing mutants will be selected on the basis of a normal progression of the Mec phenotype (10% at 3 days) and on the basis of a minimum frequency of 10 5 / mutated gamete. Below this frequency, there is a significant risk that the reverting phenotype is multi-allelic. In order to eliminate these supernumerary mutations, the selected suppressor mutants will be crossed several times with a wild line and analyzed in the heterozygous state. It may also be envisaged to cross transgenic nematodes expressing a mutated polyglutamine with a collection of mutant lines for which the mutated gene is identified by a nucleotide sequence. These are classic crosses between male animals and hermaphrodite animals. This type of collection is in development in several public and private laboratories, notably within the company EXELIXIS in San Francisco in the United States.

 The present invention therefore also relates to a method for screening therapeutic targets for the treatment of polyglutamine-expanding diseases comprising: - the determination of the mutation allowing the reversal of the neuronal dysfunction presented by the transgenic nematodes in accordance with the invention, - the identification of the gene comprising this mutation, - the search for homology of sequences with a human gene, - the determination of the function of the protein encoded by this gene.

 The determination of the mutation allowing the reversal of the neuronal dysfunction can be carried out: - in a first embodiment by:

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 - the random mutagenesis of all the genes of transgenic nematodes in accordance with the present invention, and - the isolation of said nematodes having a reversion of the phenotype of neuronal dysfunction, or - in a second embodiment by: - the crossing of transgenic nematodes in accordance the invention with nematodes belonging to mutant lines for which the mutated gene is identified by a nucleotide sequence, and - the isolation of nematodes from the above-mentioned cross showing a reversal of the phenotype of neuronal dysfunction.

 It will then be a question of characterizing these genetic suppressors in order to select those which constitute good therapeutic targets (criteria: relevance of nematode data in humans, more feasibility in vivo of modulation of activity of target proteins). The detail of research at this level can only be defined after identification of the genes containing the suppressive mutations. However, the search for sequence homologies for the genes in question in humans (for analysis of tissue expression profiles) and in other organisms (for possible analysis of biological function) will be carried out in all cases. Isolation or cloning of homologous human cDNAs and obtaining antibodies will be accomplished in order to determine the expression profile of these genes at the mRNA and protein levels. The existence of a human counterpart expressed in the brain (possibly at variable levels in HD target neurons: caudate, cortex) will be one of the essential selection criteria for a therapeutic target. The next step will be to transpose the data obtained in the nematode into HD models adapted to pre-clinical pharmacology, then possibly to humans.

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 The present invention therefore also relates to the use of a transgenic nematode in accordance with the present invention for the screening of therapeutic targets for the treatment and / or prevention of polyglutamine-expanding diseases. In particular, the determining step of the above screening will be the determination of the mutation allowing the reversal of the neuronal dysfunction as indicated above.

 A subject of the invention is also the use of the gene or the product of the gene in which the mutation has been determined for the preparation of a medicament intended for the treatment and / or prevention of polyglutamine-expanding diseases.

 Finally, the present invention also relates to the use of a transgenic nematode in accordance with the invention for the identification of compounds having an inhibiting activity on the abnormal phenotype presented by said nematode. This involves, for example, subjecting said nematode to, or bringing it into contact with, at least one compound capable of having the said inhibition activity and of regularly monitoring the effect of this contact and of this exposure on the phenotype. abnormal presented by the nematode. Obviously, the compounds giving rise to a partial or total reversion of the abnormal phenotype in question will then be characterized and will be the subject of in-depth studies and analyzes with a view to preparing a medicament intended for the treatment and / or prevention of polyglutamine expanding diseases.

PRESENTATION OF THE FIGURES
Figure 1. Diagram of transgenes
The constructs used allow the expression of fusion proteins containing from the N-terminal side to the C-terminal side a fragment of the mec-3 protein of 29 amino acids, the first 17 amino acids of the htt,

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the polyglutamine domain of variable length (18.88, or 128 Gln), the 17 amino acids of the htt according to the polyglutamine domain, and GFP (Green fluorecent protein). The expression vector used is Tu482 (Martin Chalfie, Columbia University, New-York, NY, USA), a derivative of pPD95. 65 (public access; Andrew Fire, Carnégie Institute of Washington, USA). The fragments of the htt containing 18 gln come from the Laboratory of Genomic Biology (CEPH, Paris), those containing 88 gln were obtained from plasmids coming from the laboratory of Christopher Ross (Johns Hopkins University, Baltimore USA), and those containing 128 gln were obtained from plasmids from the laboratory of Michael Hayden (University of British Columbia, Vancouver, Canada). Legend: aa: Amino acids; Polygln: polyglutamine domain
Figure 2: Polyglutamine expansion in C elegans neurons alters mechanosensation
Mechanosensation was measured in three-day-old nematodes by stimulating their body by contact with an eyelash. 20 to 30 animals were placed on standard food plates seeded with an OP50 food source and were maintained as such. The animals were transferred to fresh plates 24 h before the test to minimize the adaptive touch response effects. The defective responses for mechanosensation (MEC) were recorded for each worm plate (N = 4). A posterior MEC phenotype is defined by insensitivity to touch at the tail. An anterior MEC phenotype is defined by an insensitivity to touch at the level of the head. At the level of FIGS. 2A and 2B, a simple mechanosensory response was recorded per animal. In FIGS. 2C and 2D, 10 consecutive mechanical, 5 anterior and 5 posterior responses were recorded in 50 animals of each genotype. Thus, each animal received a score from 0 (for 10 consecutive positive responses) to 10 (for no positive response). Figure 2A: Posterior MEC as a percentage for each line of worms, with error bars indicating the standard error

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at the average for each plate test tested At least 5 independent transgenic lines for a given construct were tested The wild type N2 shows some MEC responses, such as transgenic worms for mec-3p :: GFP [GFP] and mec-3p: : Httl-57 (19Q) :: GFP [19-GFP]. The mec-3p :: Httl-57 (88Q) :: GFP lines [88Q-GFP] showed significant alterations in the posterior MEC response of between 27 and 63%. Lines of the mec-3p :: Httl-57 (128Q) :: GFP [128Q-GFP] genotype in general even showed more posterior MEC responses, between 62 and 77%. In the mec-3p :: Httl- 57STOP (128Q) :: GFP [STOP-128Q] genotype lines, the responses were not significantly different from the control values. The mec 3p lines :: Htt1-57 (128Q ) MINUSGFP [128Q MINUS GFP] showed MEC responses similar to the control lines Figure 2B percentage of the previous MEC phenotypes for the wild type N2 and mec-3p :: Htt1-57 (128Q) :: GFP (line A) [128Q- GFP], with the error bars indicating the standard error at the mean for each plate test tested Figure 2C: MEC responses from animals N2 and mec-3p :: Htt1-57 (128Q) :: GFP (line A) [128Q-GFP], with the error bars indicating the standard error as the average for each response. Figure 2D: Diffuse marking of the response in points of individual CEM of graph C, indicating the degree of overlap between the individual data of N2 (empty squares) and mec- 3p :: Httl-57 (128Q) :: GFP (line A ) [Htt (128Q) -GFP] (solid circles)
Figure 3: Representative patterns of GFP expression of transgenic fusion proteins in PLM mechanosensation neurons
The transgenic animals were washed from the food plates in M9 buffer, immobilized by incubation in levamisole, installed on 3% agarose supports on microscope slides and placed under a glass slide In all cases, cellular GFP distributions were established in PLM posterior mechanosensory neurons under a set of FITC filters Observations were made under an XI 00 objective. The images were generated with a CCD camera using Photoshop Adobe software.

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The posterior aspect is on the right in all cases. Figure 3A: mec- 3p :: GFP. GFP is diffusely expressed throughout the nucleus, cell body and axons. Rarely, bright spots of fluorescence are observed along the axonal projections. This can represent deficiencies in the axons themselves or an accumulation of proteins at specific sites. Figure 3B: mec-3p :: Httl-57 (19Q) :: GFP. GFP accumulates in two large sets, apparently around the nucleus Diffuse expression is detected along the axons with certain cytoplasmic aggregations (arrow) The GFP signal outside the image in the lower right corner of the diagram comes from the body cell from another animal PLM. Figure 3C: mec-3p :: Httl-57 (88Q) :: GFP GFP forms important perinuclear aggregates as visualized by two bright signals around the nucleus. Unlike PLM in mec- 3p :: GFP or mec-3p :: Httl-57 (19Q) animals, cytoplasmic aggregates along the axons are commonly observed (arrows). These can be considerably larger than those observed in mec- 3p neurons :: Httl-57 (19Q) :: GFP (lower right arrow) The GFP signal outside the image in the upper right corner of the diagram comes from the cell body of the other PLM of the animal Figure 3D: mec-3p :: Httl- 57 (128Q) :: GFP Intense GFP signals are observed around the nucleus, with two typical bright signals, such as in mec-3p :: Httl- 57 (88Q) :: GFP. In general, the PLM neurons of this genotype do not have a greater number of aggregates along the axons (arrows) than those in mec-3p :: Httl-57 (88Q) :: GFP. The GFP signal outside the image in the lower left corner of the diagram comes from the cell body of the animal's other PLM.

Figure 4: Representative image by confocal analysis of GFP fusion proteins in PLM neurons of transgenic lines
Animals were incubated in 150 ng / ml of DAPI in ethanol for 30 min, followed by 30 min of rehydration in M9 buffer. The worms were then briefly incubated in levamisole and installed on slides containing solidified supports of agarose at 3%

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 to prevent movement during image acquisition. Confocal sections of 0.4 microns were taken from signals around the nucleus, as detected by DAPI labeling, using UV and FITC filters for DAPI and GFP, respectively. The DAPI marking is pseudocoloured in red, with GFP in green. Z series reconstructions are shown here, where the yellow indicates a co-location of the DAPI and GFP signals. The posterior aspect is on the right in all cases. The scale: 5 microns. Figure 4A: mec-3p :: GFP. A diffuse GFP signal is apparent along the axonal projections, throughout the cell body and in the nucleus (arrow). Figure 4B: mec-3p :: Httl-57 (19Q) :: GFP. The GFP signal shows an accumulation near the nucleus, with protein deposits also present within the nucleus (arrow). Figure 4C: mec-3p :: Httl- 57 (88Q) :: GFP. The worms gave rise to a GFP signal in the form of a ring around the nucleus, with also a signal detected inside the nucleus (arrow). Figure 4D: mec-3p :: Httl-57 (128Q) :: GFP. The two PLM cores are visible. On the left, the signal is detected in the nucleus (arrow), but not in the nucleus on the right. The worms showed a significant accumulation of GFP on one side of the nucleus with numerous point deposits around the nuclear surface.

Figure 5: Diagram of the stages of the reversion tests in the C. elegans worm
Figure 5A: by random mutagenesis
Figure 5B: using collections of mutants.

 The present invention is not limited to the description above but on the contrary encompasses all the variants The experiments and results reported below serve to better understand the invention but are only mentioned for purely illustrative purposes.

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EXPERIMENTATIONS AND RESULTS
A. Generation of new mec3 transgenic lines
The original transgenic lines were all obtained from a dpy-20 mutant. However, in order to improve the efficiency of the transformation, the inventors also carried out injections into the mutant lin-15 (phenotype: several vulvae). From there, new DNA constructs, as well as the above transgenes have transformed these strains to control any potential effects of the genetic background. The sequence of all transgenes was checked before injection. During the resequencing of the original construction mec-3p :: Httl-57 (84Q) :: GFP injected with COLUMBIA, it was discovered that this transgene actually coded for a protein containing 88 contiguous glutamines and not 84 as previously postponed. The inventors have generated new constructions comprising: - 128 glutamines: mec-3p :: Htt1-57 (128Q) :: GFP, - 128 glutamines not fused to GFP, to control the GFP effects: mec-3p :: Httl-57 (128Q) MNUSGFP, - two stop codons just before the sequence coding for the expansion of 128 glutamines: mec-3p :: Httl-57STOP (128Q) :: GFP, to control the effects of messenger RNA.

 These three constructs, as well as the GFP alone, variants 19Q and 88Q, all under the control of the mec-3 promoter, were injected with wild type DNA lin-15 (phenotypic marker for selection restoring a wild phenotype lin-15 ). In each case, a minimum of 5 independent transgenic lines were selected for the following analyzes.

B. Mechanosensation analyzes
The inventors were interested in determining whether or not they could reinforce the MEC phenotype originally observed to use a suppressor screen and they examined the MEC responses in animals aged 3 days (after this age, the animals significantly less offspring). It was found that, as before, animals

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 transgenic expressing GFP alone or GFP-19Q showed a level of mechanosensation close to the wild type (Figure 2A). However, in 5 lines of GFP-88Q examined, 2 showed a significantly higher level of MEC compared to that observed previously while the 3 remaining lines showed posterior MEC phenotypes similar to those observed previously. In contrast to the variability in the effects of 88Q in the transgene, all the 128Q lines examined showed a strong MEC phenotype posterior to around 70%. The control transgenic animals expressing 128Q with 2 stop codons upstream of the polyglutamine expansion have showed a level of MEC close to the wild type. 8 transgenic lines of 128Q MINUS GFP showed a level of MEC close to the wild type. Although the inventors know from the fluorescent signal that the GFP fusion proteins have been expressed, it is possible that there is no detectable expression of 128Q proteins in these lines. The inventors commonly examine this notion of immunohistochemistry with antibodies directed against the N-terminal end of huntingtin. It is also possible that GFP may be present in PLM neurons acting as an sensitizing agent for the effects of polyglutamine expansion on MEC to be detected. This can be tested by co-injecting mec-3p :: GFP with this construction of mec-3p :: Httl-57 (128Q) MINUSGFP.

 Previous mechanosensation was tested in wild type worms and 3 day old 128Q transgenic worms. A moderate but significant MEC phenotype was detected (Figure 2B). The inventors wonder whether it might be possible to detect larger differences in mechanosensation between wild type N2 worms and 128Q worms by testing animals with multiple stimuli. They subjected the worms to 10 mechanosensory stimuli, 5 at the head and 5 at the tail and measured the responses in N2 and 128Q. A maximum MEC phenotype would therefore give a rating of 10 while 10 consecutive positive responses would give a rating of 0. In this particular case, the animals N2 were scored 1.3 while the animals 128Q were scored 5.1 (Figure 2C ). Examination of the individual point data from these tests reveals a slight overlap between N2 and 128Q (Figure

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 2D), which suggests that there is a potentially interesting way to screen for F2 suppressors of the MEC 128Q phenotype.

C. Distribution of the aggregation patterns of polyglutamines
The inventors examined the patterns of GFP expression of fusion proteins in PLM neurons (Figures 3 and 4). The mec-3p :: GFP transgenic lines showed a diffuse signal within the nucleus, the body and the cellular machinery without significant or obvious accumulation at the level of a specific cellular localization (Figures 3A and 4A). The control transgenics, mec-3p :: Httl-57 (19Q) :: GFP, have shown an intense accumulation of the GFP signal, typically visible as two diametrically opposite sets, apparently around the nucleus. Diffuse expression was observed in the nucleus (see Figure 4B) Likewise, a diffuse signal was evident along the axonal projections with a small but significant number of fusion protein aggregates (Figure 3B). In the mec-3p :: Httl-57 (88Q) :: GFP transgenes, the accumulation of proteins around the nucleus was more intense. Like the two large sets observed in the mec-3p :: Httl-57 (19Q) :: GFP lines, smaller point accumulations appeared around or near the nucleus (Figures 3C and 4C). A signal was also detected in the nucleus Along the axonal projections, several aggregates were generally visible. Diffuse expression was not detected (Figure 3C). The mec-3p :: Httl-57 (128Q) :: GFP transgene also showed significant accumulations near or around the nucleus. These signals had a more fragmented appearance than in other genotypes. In addition, GFP nuclear signals appeared less ordinary than in other lines (Figure 4D) As was the case with mec-3p :: Httl-57 (88Q) :: GFP, a diffuse signal did not was detected along the axonal projections. However, there appeared to be a greater number of aggregates along the axons of mec-3p :: Httl-57 (128Q) :: GFP (Figure 3D). Under a confocal microscope, aggregation patterns around or near the nucleus in the mec-3p transgenes :: Httl-57 (88Q) :: GFP and mec-3p :: Httl-57 (128Q) :: GFP are reminiscent with the appearance of the Golgi apparatus. The huntingtin protein has recently been shown

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was located near this structure (Vélier J et al., 1998). Transport via the Golgi apparatus could be a source of cellular disorganization in the inventors' system
From analyzes of the cellular distribution of GFP, the inventors concluded that the polyglutamine expanding fusion proteins formed aggregates, as in other model systems (Davies SW et al., 1997; Scherzinger E et al., 1997; Martindale D et al., 1998; Cooper JK et al., 1998; LI SH & Li XJ, 1998) and human diseases (DiFiglia M et al., 1997; Maat-Schieman ML et al., 1999, Sapp E et al., 1999). In any event, there appears to be an inverse correlation between the length of polyglutamine expansion and the presence of nuclear proteins. However, there appears to be a moderately well correlated relationship between the number of cytoplasmic aggregates and the size of the polyglutamine expansion. Thus, the relationship between aggregation and pathology remains an open question (Kuemmerle S et al., 1999).

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Claims (26)

1. Transgenic nematode with a phenotype of neuronal dysfunction in the absence of neuronal death in the young adult stage.  2. Transgenic nematode according to claim 1, characterized in that the phenotype of neuronal dysfunction has a penetrance of at least 50%, preferably at least 70%.  3. Transgenic nematode according to claim 1 or 2 comprising a construct allowing the expression of a protein having a polyglutamine domain under the control of a promoter specific for neurons.  4. Transgenic nematode according to claim 3, characterized in that the promoter is specific for touch receptor neurons.  5. Transgenic nematode according to claim 4, characterized in that it is the promoter of the mec-3 gene.  6. Transgenic nematode according to one of claims 3 to 5, characterized in that the protein having a polyglutamine domain is an N-terminal fragment of huntingtin.  7. Transgenic nematode according to claim 6, characterized in that the polyglutamine domain comprises between 39 and 128 glutamine residues, preferably between 88 and 128 glutamine residues and more preferably 128 glutamine residues.  8. Transgenic nematode according to one of claims 3 to 7, characterized in that the protein is a fusion protein.  9. Transgenic nematode according to claim 8, characterized in that the fusion protein comprises a protein having a <Desc / Clms Page number 38>  polyglutamine domain and a protein allowing the visualization of the expression of the fusion protein.  10. Transgenic nematode according to claim 9, characterized in that the protein allowing the visualization of the expression of the fusion protein is the green fluorescent protein (Green Fluorescent Protein or GFP).  11. Transgenic nematode according to claim 10, characterized in that the construction comprises a nucleic sequence coding for the first 29 amino acids of the protein Mec-3, then coding for the first 17 amino acids of huntingtin, then coding for the polyglutamic domain comprising from 39 to 128, preferably 88 to 128, more preferably 128 glutamine residues, then coding for the 17 amino acids of huntingtin following the polyglutamine domain, then for GFP, under the control of the promoter of the mec-3 gene .  12. Transgenic nematode according to one of claims 1 to 11, characterized in that it is Caenorhabditis elegans.  13. Transgene comprising a nucleic sequence comprising the nucleic sequence of a promoter specific for neurons affected by the affected organism operably linked to the nucleic sequence of a gene coding for a protein having a polyglutamine domain.  14. Transgene according to claim 13, characterized in that the promoter is the promoter of the mec-3 gene.  15. Transgene according to claim 13 or 14, characterized in that the nucleic sequence of the gene coding for the protein is itself operably linked to the nucleic sequence of a protein allowing the visualization of the resulting fusion protein. <Desc / Clms Page number 39>  16. Transgene according to one of claims 13 to 15, characterized in that the protein having a polyglutamine domain is huntingtin 17. Transgene according to claim 16, characterized in that the polyglutamine domain comprises between 39 and 128 glutamine residues, preferably between 88 and 128 glutamine residues and more preferably 128 glutamine residues.  18. Transgene according to claim 17, characterized in that it comprises a nucleic sequence coding for the first 29 amino acids of the protein Mec-3, then for the first 17 amino acids of huntingtin then coding for the polyglutamine domain comprising from 39 to 128, preferably 88 to 128, more preferably 128 glutamine residues, then for the 17 amino acids of huntingtin following the polyglutamine domain, then for GFP, under the control of the promoter of the mec-3 gene.  19. Use of a transgene according to one of claims 13 to 18 to transform a nematode, preferably C. elegans.  20. A method of screening therapeutic targets for the treatment of polyglutamine-expanding diseases comprising: - the determination of the mutation allowing the reversal of the neuronal dysfunction presented by the transgenic nematodes according to one of claims 1 to 12, - the identification of the gene comprising this mutation, - the search for sequence homology with a human gene, - the determination of the function of the protein encoded by this gene.  21. Screening method according to claim 20, characterized in that the determination of said mutation comprises: <Desc / Clms Page number 40>  - the random mutagenesis of all the genes of transgenic nematodes according to one of claims 1 to 12, and - the isolation of said nematodes having a reversion of the phenotype of neuronal dysfunction.  22. Screening method according to claim 20, characterized in that the determination of said mutation comprises: - the crossing of transgenic nematodes according to one of claims 1 to 12 with nematodes belonging to mutant lines for which the mutated gene is identified by a nucleotide sequence, - the isolation of nematodes from the above cross showing a reversal of the phenotype of neuronal dysfunction.  23. Use of a transgenic nematode according to one of claims 1 to 12 for the screening of therapeutic targets for the treatment and / or prevention of polyglutamine-expanding diseases.  24. Use according to claim 23 comprising the determination of the mutation allowing the reversal of the neuronal dysfunction.  25. Use of the gene or gene product in which the mutation has been determined according to claim 24 for the preparation of a medicament intended for the treatment and / or prevention of polyglutamine-expanding diseases.  26. Use of a transgenic nematode according to one of claims 1 to 12 for the identification of compounds having an inhibiting activity on the abnormal phenotype presented by said nematode.