FR2951083A1 - USE OF A DNA REPLICATION INHIBITOR FOR THE TREATMENT OF POLYGLUTAMINE EXPANSION NEURODEGENERATIVE DISEASES - Google Patents

Abstract

The present invention relates to the use of a DNA replication inhibitor for the treatment of a neurodegenerative disease by polyglutamine expansion.

Description

The present invention relates to the field of medicinal products intended for the treatment of neurodegenerative diseases by polyglutamine expansion, more particularly dominant spinocerebellar ataxias. BACKGROUND OF THE INVENTION The present invention relates to the field of medicaments for the treatment of neurodegenerative diseases by polyglutamine expansion, more particularly to dominant spinocerebellar ataxias. Human neurodegenerative diseases represent a major public health problem given the aging of the population. Indeed, many of these diseases have an increased incidence with age. This could be related to the failure, with age, of adaptive mechanisms capable of containing the cellular dysfunctions at the origin of these pathologies. Among the neurodegenerative diseases, the dominant autosomal spinocerebellar ataxias (ADCA, also known as spinocerebellar dominant ataxias) represent a group of more than twenty rare diseases (1 in 10 000 is affected), characterized by motor disorders related to degeneration of the cerebellum. The most common dominant spinocerebellar ataxias (SCA1, 2, 3, 6, 7, 17) belong to the class of polyglutamine expansion diseases, which also include Huntington's disease, dentato-rubro-pallido-luysian atrophy ( DRPLA) and Kennedy's disease (or spino-bulbar muscular atrophy, SBMA) (Soong and Paulson 2007, Gatchel and Zoghbi 2005). These diseases are due to an expansion mutation of the CAG codon (coding for glutamine) in the gene sequence coding for the protein responsible for the disease (eg, Huntingtin, Atrophin-1, Ataxin-1, 2, 3 or 7). . Beyond a number of repetitions of glutamine, the protein becomes toxic to the cell and the body, according to mechanisms still poorly understood. Many animal models have been developed to study these human pathologies. Of these, invertebrate models (Drosophila and nematode) have been used to study polyglutamine expansion diseases and Parkinson's and Alzheimer's diseases. In fact, these models reproduce the main characteristics of human pathologies and make it possible to carry out rapid genetic screens (Bilen and Bonini 2005, Zoghbi and Botas 2002, Bonini

and Fortini, 2003). The functional conservation of the different regulatory pathways between invertebrates and vertebrates makes it possible to use the results obtained to develop treatments for human pathologies. Thus, several compounds that have been shown to be effective in Drosophila are in clinical trials in humans (Shulman et al., 2003). Since most neurons do not divide in the adult brain, the cell cycle markers characteristic of the DNA replication phase are not normally activated. However, it has been shown in models of neurodegenerative diseases such as Parkinson's disease, amyotrophic lateral sclerosis, tauopathies and Alzheimer's disease, which are not polyglutamine expansion diseases, that post-mitotic neurons. abnormally re-express many cell cycle markers, such as DNA polymerases a, 8, 13 and primase, and replicate their DNA (Herrup et al., 2004; Lee et al., 2003; Ranganathan and Bowser, 2003, Ueberham and Arendt, 2005, Andorfer et al., 2005, Copani et al., 2002). For example, Khurana et al. Showed in a Drosophila model of tauopathy that the cell cycle is reactivated in post-mitotic neurons of adult flies, and that inhibition of certain cell cycle proteins is able to suppress the phenomenon. observed neurodegeneration of the eye in this pathology (Khurana et al., 2006 and 2007). With regard to Alzheimer's Disease, Copani et al. Have shown, in vitro, using a model of cells expressing amyloid A peptide (342, that the toxicity of this peptide is reduced in cells where DNA polymerase beta is inactivated (Copani et al., 2002, 2006, 2007 and 2008).

In contrast, for neurodegenerative diseases by polyglutamine expansion, including dominant spinocerebellar ataxias, Khurana et al. (2006) showed using a Drosophila model of tauopathy, that the expression of the protein MJD-78Q (MJD protein, also called ATXN3, with an expansion of 78 glutamines) induces apoptotic neurodegeneration, without the authors have observed the presence of certain markers of replication and mitosis (PCNA, PH3) or have managed to

modulate the pathology by inhibiting the proteins involved in the cell cycle in this model. There is currently no cure for polyglutamine expansion diseases. However, clinical trials are ongoing with, in some cases, molecules that have shown efficacy on Drosophila models. For example, inhibitors of histone deacetylase 8 (HDAC8, which plays a role in the regulation of gene expression and DNA repair, International Application WO 2007/130419) and inhibitors of the protein Hsp9O ("chaperone" protein, which plays a role in protecting the three-dimensional folding of proteins), such as derivatives of geldanamycin. There is therefore a need for novel therapeutic targets as well as new pharmaceutical compositions useful for the treatment of polyglutamine expansion diseases. The inventors have established three Drosophila models of dominant spinocerebellar ataxias (SCA1, SCA3 and SCA7). The principle of these models is shown in Figure 1: the polyglutamic pathological forms of the ATXN1, ATXN3 and ATXN7T proteins involved in human ADCA are induced in adult Drosophila (in the eye, neurons or glial cells) using the two-component system GAL4-UAS (Brand and Perrimon, 1993, Latouche et al., 2007). In the SCA1 model, the protein Ataxin 1 (ATXN1) comprising an expansion of 82 glutamines (ATXN1-82Q) was expressed. In the SCA3 model, the protein Ataxin 3 (ATXN3) comprising an expansion of 70 glutamines (ATXN3-70Q) was expressed. In the SCA7 model, the truncated Ataxin 7 protein (ATXN7T) comprising an expansion of 102 glutamines (ATXN7T-102Q, Latouche et al., 2007) was expressed. For the neuronal expression of pathological proteins, the inventors have used an elav-GAL4GS line for which the transcriptional activity of GAL4 is under the control of an inducer, RU486 (mifepristone), which can be ingested in adult flies. (Latouche et al., 2007); this makes it possible to overcome the effects on the development of Drosophila that could have these proteins or the different products tested in the sieves, and to follow precisely in the adult fly the development of the pathology. These Drosophila models have

were then used to identify (screen), by inactivation by interfering RNA, genes capable of modulating these pathologies. During these screens, the inventors have surprisingly identified the gene DNA pol-alpha50, orthologue of the human PRIM1 gene (coding for the primase, a protein that plays a major role in the replication of DNA) as a major modulator of these 3 ataxies. In fact, the inventors have shown that the reduction of the level of the DNApo1-alpha50 protein by RNA interference makes it possible to largely eliminate the toxic effects of the pathological proteins in the 3 Drosophila models SCA1, SCA3 and SCAT studied. The observed cell protection concerns both neurons and glial cells. Other genetic screens on the Drosophila SCA3 model have allowed the inventors to identify several other actors of DNA replication capable of modulating the SCA3 pathology: they are the mcm2 and dup / Cdtl genes, necessary for replication. DNA, whose inactivation rescues the pathological phenotype, the Huwel gene, a DNA replication inhibitor whose gain in function has a protective effect in SCA3 pathology, and grp / Chk1, coding for a major kinase of the ATR pathway ("Ataxia Telangiectasia and Rad3-related") involved in the control of DNA replication, whose gain of function also saves the pathological phenotype.

These results show that several actors (genes, proteins) playing a role in DNA replication and its control appear as potential therapeutic targets in neurodegenerative diseases by polyglutamine expansion: - the primase (DNApolalpha50 / PRIM1) ( Arezi and Kuchta, 2000, Lao-Sirieix et al., 2005 and Muzi-Falconi et al., 2003), which intervenes during phase S of the cell cycle which precedes later phases of the cycle (G2, M, GI) ( Sclafani and Holzen, 2007); the DNA replication complexes comprising the ORC and MCM proteins, which are put in place during the pre-replication phase preceding the replication of the DNA proper. Activation of these complexes induces replication, in which the primase synthesizes the RNA primers

necessary for DNA polymerases (polalpha and epsilon) to effect the synthesis of a complementary DNA strand; proteins involved in DNA repair mechanisms when DNA replication is blocked, such as ATM ("ataxia telangiectasia mutated") and ATR ("ATM and Rad3-related") kinases (Cimprich and Cortez , 2008); proteins controlling the course of the cell cycle, such as cdk kinases and cyclins (Sclafani and Holzen, 2007). The present invention therefore relates to the use of a DNA replication inhibitor for the preparation of a pharmaceutical composition for the treatment of a neurodegenerative disease by polyglutamine expansion. By DNA replication inhibitor is meant a compound which, when administered to a subject, inhibits totally or by at least 20%, and in order of increasing preference by at least 50%, % and 95%, the replication of DNA in tissues, preferably neuronal and / or glial cells, of said subject compared to a level of DNA replication measured in the absence of administration (ie, without treatment) of said inhibitory agent. Inhibition of DNA replication can be determined by techniques known to those skilled in the art, such as, for example, in vitro, by the incorporation of 3H-labeled thymidine or bromodeoxyuridine (BrdU; thymidine) or flow cytometric analysis or, in vivo, through the analysis of the final size of rapidly growing organs (Shibutani et al., 2008). The DNA replication inhibitor can be a chemical or biological compound of natural or synthetic origin. It may be chosen from an antibody, a nucleic acid (such as an interfering RNA, an antisense RNA or a ribozyme), a peptide, a protein, preferably an interfering RNA. Inhibition of DNA replication is of course dependent upon the doses at which the inhibitor is used. The DNA replication inhibitor may also have one or more other actions that are not inhibiting the replication of the DNA. However, the inhibitory action of DNA replication

of that compound must be preponderant to the dose (or concentration) used in relation to its other actions. According to a preferred embodiment of the invention, said inhibitor of DNA replication is an inhibitor of a protein or a protein complex involved in the replication of DNA, chosen from the following factors: initiation of replication, helicases, primases, DNA polymerases, DNA ligases, topoisomerases, kinases, nuclear proliferation nuclear antigens (PCNA), replication factors A and C, preferably a primase. The inhibitor may act by preventing the attachment of a ligand (e.g., substrate) to the active site of the protein, or cause conformational deformation of the protein that renders it inactive. According to another preferred embodiment of the invention, said inhibitor of DNA replication inhibits the expression of a gene selected from the prim1 genes (coding for the primase DNA), mcm2 (coding for the protein "minichromosome maintenance complex component 2"), Cdtl (coding for the "chromatin-licensing and DNA replication factor 1" protein) and the orthologous genes of these genes, preferably the priml gene. According to another preferred embodiment of the invention, said inhibitor of DNA replication activates or increases the expression of a DNA replication inhibiting gene selected from the Huwel genes (coding for the protein "HECT, UBA and WWE domain containing 1", Chk1 (coding for CHK1 kinase) and the orthologous genes of these genes.Inhibition of DNA replication can also be achieved with an alkylating agent (eg, cyclophosphamide platinum salts), an intercalating agent (eg, anthracyclines) or a nucleic acid analogue (eg, 5-FU). According to an advantageous embodiment of the invention, said inhibitor of DNA replication is Hydroxyurea (CH4N2O2) A mammal, preferably a human, is defined as a subject In accordance with another preferred embodiment of the invention, said polyglutamine-expanding neurodegenerative disease is selected from Huntington's disease, spinocerebellar ataxias.autosomal dominant 1, 2,

3, 6, 7 and 17, dentato-rubro-pallido-luysian atrophy and Kennedy's disease (or spino-bulbar amyotrophy), preferably autosomal dominant spinocerebellar ataxias, and more preferably autosomal dominant spinocerebellar ataxias 1, 3 and 7.

The modes and routes of administration of the pharmaceutical composition may be adapted by those skilled in the art depending on the subject, the pathology to be treated and the inhibitor of the replication of the DNA used. By way of example, the pharmaceutical composition prepared according to the invention can be formulated for oral or nasal administration, or by intravenous, intramuscular, subcutaneous, nasal or intracranial injection. Determination of the dose at which said inhibitor of DNA replication is used can be carried out by techniques known to those skilled in the art, for example in clinical trials. This dose will depend on various factors including DNA replication inhibitor activity, mode of administration, duration of administration, level of DNA, duration of treatment, other drugs or compounds used in combination with the DNA replication inhibitor, age, sex, weight, general health status and of the prior medical history of the subject being treated. By way of example, this dose may be between 5 mg / kg and 60 mg / kg, preferably between 10 mg / kg and 30 mg / kg. The pharmaceutical composition may comprise at least one pharmaceutically acceptable excipient which may be any excipient known to those skilled in the art. The pharmaceutically acceptable excipients vary depending on the DNA replication inhibitor and the mode of administration chosen. By treatment is meant curative, symptomatic or preventive treatment. The DNA replication inhibitors used in accordance with the present invention may be used in a subject with a reported polyglutamine expansion neurodegenerative disease, including the early and / or late stages of the disease. These inhibitors of DNA replication do not necessarily cure the subject with the disease, but can

delay or slow progression or prevent further progression of the disease, thereby improving the subject's condition. These DNA replication inhibitors may also be administered to a subject who does not have symptoms of a neurodegenerative disease by polyglutamine expansion, but who may develop the disease, or who has a significant risk of developing the disease. The present invention also relates to a method for treating a neurodegenerative disease by polyglutamine expansion, comprising the administration of an effective amount of at least one inhibitor of the replication of the DNA as defined above, to a subject in need of this treatment.

Of course, the specifications described above also apply to this aspect of the invention. By an effective amount is meant an amount of DNA replication inhibitor to produce the desired biological result. In addition to the foregoing, the invention further comprises other arrangements, which will emerge from the experimental examples below, as well as the accompanying drawings, in which: - Figure 1 represents the principle of the establishment of a model Drosophila expressing a human pathological ATXN protein under the control of an inducible promoter (elav). Drosophila carrying a transposon containing mutated human ATXN protein under the control of UAS regulatory sequences are crossed with individuals carrying a transposon containing the GAL4GS regulatory protein under the control of an inducible specific tissue promoter (here the neuronal promoter elav) . In the offspring, the GAL4GS protein is produced in the tissue under consideration. It only becomes active in the presence of RU486 which is brought into the animals' food. In this case, GAL4GS binds to the UAS sequences and activates the transcription of the adjacent gene and the production of the pathological human protein. The phenotypes of these individuals can then be studied; - Figure 2 represents the principle of a genetic screen by inactivation by interfering RNA. Drosophilae expressing a pathological protein (e.g., Ataxin 3) in a specific tissue (e.g., neurons) are

crossed with individuals carrying a tandem inverted repetition of a portion of the cDNA of a gene of interest modifier (noted GM). In the offspring, in the presence of RU486, Drosophila express the pathological protein and a double-stranded RNA corresponding to the GM sequences. By interference RNA phenomenon, this GM-specific double-stranded RNA (RNAi), which causes the degradation of endogenous GM RNA and therefore the decrease of the corresponding protein encoded by GM. We can then study in these individuals if the decrease in the expression of GM modifies the phenotype caused by the expression of the pathological protein; - Figure 3 represents the distribution of average lifetimes observed in the sieve of the lines from the VDRC storage center corresponding to Table II below (solid bars). The normal distribution corresponding to the same average value and the same standard deviation is represented in empty bars. The position of individuals of genotype elavGS> ATXN3-70Q / DNApoI-alpha50 {v43870} is indicated by an arrow; 4: graphs showing the survival of Drosophila expressing in the neurons the pathological proteins ATXN3-70Q (a), ATXN7T-102Q (b) or ATXN1-82Q (c) is strongly reduced (closed circles) compared to that of the individuals controls with the same genotype but not expressing the pathological protein (open circles) or expressing a non-pathological protein such as EGFP or luciferase (open squares). The simultaneous reduction of the level of the primase (DNApolalpha50) (solid triangles) significantly improves survival in these three models of ataxia; FIG. 5 are photographs showing, in Drosophila, the neurodegeneration induced by the expression in the eye of the truncated pathological Atxn3 protein (photo of the medium) compared to the phenotype of a normal eye (left-hand photo) and phenotype restored by decreased expression of primase induced by interfering RNA (right picture); - Figure 6 represents a Western blot analysis of male Drosophila expressing the ATXN3-70Q pathological protein in neurons. This analysis shows that the expression of ATXN3-70Q is not modified by the presence of the DNApo1-a50- {v43870} transposon. Genotypes: elav-GAL4GS, UAS-ATXN3-

70Q / UAS-EGFP (left track); elav-GAL4GS, UAS-ATXN3-70Q / DNApo1-alpha50 {v43870} (right track); - Figure 7 is a graph showing that the survival of Drosophila expressing in a subset of glial cells ATXN7T-102Q pathological protein is greatly reduced (closed circles) with a lifetime not exceeding 16 days. The simultaneous reduction of the level of the primase (DNApolalpha50) (solid triangles) improves the survival in this glial model of ataxia. EXAMPLES 1) Materials and Methods Drosophila strains and culture Three neuronal models of different spinocerebellar ataxias (SCA1, SCA3 and SCAT) were generated by combining a pilot line (driver) expressing the GAL4 protein under the control of the specific promoter of differentiated neurons. elavGS (Latouche et al., 2007) and transposon-containing lines (pUAS, Brand and Perrimon, 1993) comprising a sequence coding for a pathological ATXN protein (pUAST-ATXN). The lines thus generated are noted elavGS> ATXN. The principle of establishing a Drosophila model expressing a human pathological ATXN protein under the control of an inducible promoter is shown in Figure 1.

The pUAST-ATXN1-82Q line (Fernando-Funez et al., 2000) expresses Ataxin 1 containing 82 glutamine repeats. The pUAST-ATXN3-70Q line was produced by transgenesis after cloning into a pUAST vector of a cDNA coding for Ataxin 3 and containing 70 repeats of the CAG triplet (Mueller et al., 2009).

The pUAST-ATXN7T-102Q line is described in Latouche et al., 2007. The pUAST-ATXN7T-102Q line was recombined with the Eaat1-GAL4 line (Rival et al., 2004), expressing the protein of interest in a subset of glial cells, to establish a glial model of spinocerebellar ataxia.

The resulting line is denoted ATQN7T-102Q.

The pUAST-ATXN3T-78Q line (Warrick et al., 1998) was crossed with the GMR-GAL4 line (accession number FBrf0091569 in the FlyBase database), expressing the protein of interest in the cells of the eye, to establish a model of spinocerebellar ataxia eye. The resulting line is noted GMR> ATXN3T-78Q. The RNAi lines (expressing an interfering RNA specific for a modifying gene of interest) used for genetic screens were obtained from the Vienna Drosophila RNAi Center (VDRC, Vienna, Austria). The other mutant lines used for genetic screens were obtained from the Bloomington Drosophila Stock Center (BDSC, Indiana University, Bloomington, USA). Figure 2 represents the principle of a genetic screen by inactivation by interfering RNA. The UAS-GFP-IR line has been described by Roignant et al., 2003. Drosophila were raised on a nutrient medium containing 8.2% yeast, 3.4% corn flour, 5% sucrose, , 1% agarose and 2.5% methyl benzoate (an antifungal). In order to allow the expression of the transgenes by the inducible elavGS> ATXN lines, the inducer, RU486 (diluted in 100% ethanol), was added to 200 μg / ml of medium (the medium supplemented with RU486 is called RU200 ).

The control medium without inductor was supplemented with the same volume of 100% ethanol. Longevity screens Femino drosophila elavGS> ATXN were crossed with males of lines bearing the mutations or transposons to be tested as well as possibly with control lines (UAS-EGFP, UAS-GFP-IR and UAS-Luci) of which expression does not change longevity. The resulting individuals were separated in groups of 30 into tubes containing an inducible nutrient medium (RU200) or containing a non-inducible medium (RUO). The screen was held at a constant temperature (26 ° C). Unless otherwise specified, the animals analyzed are males.

Individuals were transferred to a fresh medium every 2 days and dead individuals were counted. The longevity curves were compared to a

control curve by Log-Rank analysis (Peto and Peto, 1972). The control curve corresponds to the average of the longevities of the lines after elimination of the extremes of the life distribution (deciles 3 to 8) when a large number of identical genetic background lines are analyzed simultaneously (as for the RNAi line tests). from the VDRC). Alternatively, the curves corresponding to the survival of the control lines (UAS-EGFP, UAS-GFP-IR and UAS-Luci) measured under the same conditions were used. Analysis of the phenotype of the eye Females of genotype GMR> ATXN3-78Q and males of genotype DNApo1-alpha50 {v43870} and UAS-GFP-IR were raised at a temperature of 23 ° C. and crossed together. Optical examination of the structure of the eye was performed on males obtained, aged 3 days, with a Leica M205FA stereomicroscope. Western Blot and Immunodetection of Ataxin 3 Expression of Ataxin 3 in Drosophilae of the genotype elavGS> ATXN3-70Q / UAS-EGFP and elavGS> ATXN3-70Q / DNApoI-alpha50 {v43870} grown for 4 days on medium inducible was analyzed by Western blot. 10 flies of each condition (induction or non-expression of the pathological protein) were milled in 1001 μl of urea buffer supplemented with antiproteases. After centrifugation for 10 minutes at 12000 rpm at 4 ° C., the supernatants were denatured for 5 minutes at 95 ° C. in Laemmli buffer before being deposited on a 12% polyacrylamide gel, for analysis by electrophoresis. After transfer of the proteins to a nitrocellulose membrane, it was incubated overnight at 4 ° C. in 0.1% TBS-tween and 5% milk with the primary antibody (Chemicon-MAB5360) directed against Ataxine 3, diluted to 1/2000. Incubation with the secondary anti-mouse antibody antibody coupled to peroxidase (HRP) was carried out for 1h at room temperature. The revelation was performed with a luminescent substrate (Millipore). The signal was detected and quantified using a LAS-400 (Fujifilm) camera. Pharmacological tests The pharmacological tests were carried out with an antireplicative compound, hydroxyurea (Sigma). Hydroxyurea has been added to different

concentrations in the nutrient medium supplemented with RU486. The tubes were changed every two days and the longevity analyzed. The genotypes used are elavGS> ATXN3-70Q-WT2 / elavGS> ATXN3-70Q-WT2 (test1) and elavGS> ATXN3-70Q-WT2 / ATXN3-70Q-WT3 (test2), where WT2 and WT3 denote 2 different transgene insertions pUAST-ATXN3-70Q. 2) Results 2.1) Characterization of Drosophila models of spinocerebellar ataxia SCA1, SCA3 and SCAT.

Drosophila models of spinocerebellar ataxia SCA1, SCA3 and SCAT have been generated and characterized by various studies: analysis of phenotypes induced by different pilot lines ("drivers") GAL4, reduction of longevity caused by the expression of the pathological protein in the brain, labeling of brains in toto or cryostat frozen brain sections with antibodies directed against proteins of interest (ubiquitin, proteasome subunits, transcription factors) identified in mammals aggregates containing pathological proteins (Sang and Jackson, 2005), kinetic study of aggregate formation (Latouche et al., 2007), quantification of locomotor activity. Significantly, Drosophila models reproduce the characteristics of human pathologies, including a sharp reduction in life span and a progressive motor deficit. The effect on the lifespan of Drosophila obtained after crossing with different control lines and expressing pathological Ataxin 3 is given in Table I below. A decrease in the order of 35% of the longevity of individuals expressing pathological Ataxin 3 compared with individuals of the same uninduced genotype (with RU486), which does not express pathological Ataxin 3, was observed. Table I: Drosophila males of several control lines (wild (w), UAS-EGFP, UAS-GFP-IR and UAS-Luci) were crossed with females of genotype elavGS> ATXN3-70Q or elavGS (controls). The descendants of these crosses, whose genotype is indicated in column 1, were placed under induced conditions (the flies are placed on a medium containing 200 μg / l of RU486, RU200 medium) or in uninduced condition (column 2). The average lifetimes in days (column 4) were compared. A 35% decrease in life is observed when pathological Ataxin 3 is expressed, with highly significant statistical differences between the longevity curves analyzed by LogRank test (column 6). Drosophila controls not expressing Ataxin 3 are not sensitive to RU486 treatment. The numbers analyzed (N) for each condition (induced or uninduced expression) are shown in column 3. Table I: Effect on the lifespan of Drosophila obtained after crossing with different control lines and expressing Ataxin 3 pathological Genotype Condition N Ratio duration duration Life test LogRank mean average induced / uninduced / uninduced induced UAS-Stinger / elavGS No Induced 58 47.0 0.94 -3.9E-07 UAS-Stinger / elavGS Induced 137 44, 4 w / elavGS Not Induced 27 44.9 1.02 3.2E-01 w / elavGS Induced 241 45.7 UAS-EGFP / elavGS Induced 144 43.7 UAS-Luci / elavGS Induced 70 47.0 all males / elavGS 45.0 UAS-Stinger / elavGS> ATXN3-70Q No Induced 84 46.9 0.77 4.5E 15 UAS-Stinger / elavGS> ATXN3-70Q Induced 160 36.2 w / elavGS> ATXN3-70Q No Induced 58 49.4 0.65 -9.1E-26 w / elavGS> ATXN3-70Q Induced 206 32.3 UAS-EGFP / elavGS> ATXN3-70Q No Induced 60 49.1 0.69 -9.2E-30 UAS- EGFP / elavGS> ATXN3-70Q Induced 182 33.8 GFP IR / elavGS> ATXN3-70Q No Induced 27 4 4.1 0.62 -3.7E-17 GFP IR / elavGS> ATXN3-70Q Induced 119 27.3 UAS-luci / elavGS> ATXN3-70Q No Induced 24 48.8 0.46 -2.6E-15 UAS -luci / elavGS> ATXN3-70Q Induced 52 22.4

2.2) Suppression of Toxicity in Drosophila Models of Spinocerebellar Ataxia (SCA1, SCA3 and SCAT) by Primase Inactivation 98 Interfering RNA-expressing Lines, from the VDRC Storage Center, were analyzed for their ability to modify the lifespan of Drosophila co-expressing pathological Ataxin 1, 3 or 7 respectively and RNAi. For this, females of the genotype elavGS UAS-ATXN1-82Q, elavGS UASATXN3-70Q and elavGS UAS-ATXN7T-102Q were respectively crossed with males of different genotypes and the offspring was analyzed (according to the principle of genetic screen represented at Figure 2).

The results for the Drosophila SCA3 model are shown in Table II below and in Figure 3: a shorter life span was observed for most individuals expressing pathological Ataxin 3 (Table II), with a distribution of this life close to a normal distribution (Figure 3).

Table II: Screening results of genes modifying the toxicity of Ataxin 3 on the Drosophila SCA3 model after induction (on RU200 medium). The genotypes are carried in column 2, the numbers (N) in column 3 and the lifespan in column 4. The control curve corresponds to the average of the lines after elimination of the extremes of the life distribution (deciles 3 to 8 ). For each genotype, the ratio of average life time to that of the control curve is reported in column 5 and the probability of a significant difference between the 2 survival curves by a LogRank test is given in column 6 An increase in the lifetime of 44% is observed when the DNApo1-alpha50 is inactivated (second last line of the table) .25 Table II Gene Genotype N duration of Ratio Test of life duration of LogRank average life after average elimination / curve control deciles 3 to 8 T-cpl T-cpl {v34070} / elavGS> ATXN3-70Q 93 13.5 0.54 -6.4E-73 1 (2) 05070 1 (2) 05070 {v35923} / elavGS> ATXN3-70Q 91 20.0 0.80 -1.4E-21 CG14542 CG14542 {v24869} / elavGS> ATXN3-70Q 92 20.8 0.83 -1.7E-13 eIF5 eIF5 {v29070} / elavGS> ATXN3- 70Q 104 21.0 0.84 -2.7E-12 ashl ashl {v28982} / elavGS> ATXN3-70Q 99 21.5 0.86 -3.7E-11 Rpn6 Rpn6 {v18022} / elavGS> ATXN3-70Q 96 21.5 0.86 -2.9E-22 CG14224 CG14224 {v47447} / elavGS> ATXN3-70Q 92 21.7 0.86 -2.5E-09 CG2051 CG2051 {v 33459} / elavGS> ATXN3-70Q 83 21.7 0.86 -3.6E-07 Prosbeta3 Prosbeta3 {v31608} / elavGS> ATXN3-70Q 90 21.7 0.87 -2.7E-10 G9a G9a {v25474} lei avGS> ATXN3-70Q 100 21.8 0.87 -2.2E-09 1 (1) G0148 1 (1) G0148 {v24184} / elavGS> ATXN3-70Q 74 22.0 0.88 -2.4E- 01 flow not {v45776} / elavGS> ATXN3-70Q 80 22.0 0.88 -2.3E-06 Ada2b Ada2b {v24076} / elavGS> ATXN3-70Q 90 22.1 0.88 -1.7E-12 in in {v11322} / elavGS> ATXN3-70Q 81 22.1 0.88 -3.2E-10 Hsp70Aa Hsp70Aa {v41748} / elavGS> ATXN3-70Q 97 22.9 0.91 -5.6E-06 Rtfl Rtfl { v27341} / elavGS> ATXN3-70Q 90 22.9 0.91 -1.6E-03 alphaTub84B alphaTub84B {v33427} / elavGS> ATXN3-70Q 99 23.0 0.92 -2.7E-03 CG4165 CG4165 {v41976} / elavGS> ATXN3-70Q 93 23.2 0.93 -2.7E-06 CG14712 CG14712 {v18347} / elavGS> ATXN3-70Q 98 23.2 0.93 -3.2E-03 Case Case {v12648} / elavGS > ATXN3-70Q 96 23.3 0.93 -1.9E-03 SmD3 SmD3 {v35934} / elavGS> ATXN3-70Q 85 23.5 0.94 -1.3E-03 CG6905 CG6905 {v13492} / elavGS> ATXN3 -70Q 92 23.5 0.94 -1.0E-02 Usp36 Usp36 {v11152} / elavGS> ATXN3-70Q 91 23.6 0.94 -6.4E-04 Taft Tafl {v41099} / elavGS> ATXN3-70Q 90 23.6 0.94 -1.0E-03 HDA C4 HDAC4 {v20522} / elavGS> ATXN3-70Q 89 23.7 0.95 -2.6E-05 cdk7 cdk7 {v10442} / elavGS> ATXN3-70Q 88 23 , 8 0.95 -6.8E-01 sop sop {v20963} / elavGS> ATXN3-70Q 87 23.8 0.95 -1.6E-01 Pplalpha-96A Pplalpha-96A {v27673} / elavGS> ATXN3-70Q 91 23.8 0.95 -1.6E-04 CG4049 CG4049 {v6981} / elavGS> ATXN3-70Q 80 23.9 0.95 2.2E-02 CG33182 CG33182 {v46444} / elavGS> ATXN3-70Q 100 24, 0 0,96 -2,2E-03 Epac Epac {v50373} / elavGS> ATXN3-70Q 81 24.0 0.96 -2.2E-01 PICK1 PICK1 {v22269} / elavGS> ATXN3-70Q 91 24.1 0 , 96 -2.5E-01 Mi-2 Mi-2 {v10766} / elavGS> ATXN3-70Q 90 24.1 0.96 -5.5E-03 Sirt2 Sirt2 {v21999) / elavGS> ATXN3-70Q 86 24, 1 0,96 -9,1E-03 enc enc I (vI5291) / elavGS> ATXN3-70Q 85 24,2 0,97 -6,0E-02 4406-1-1M} UAS-4406-1-1M} / elavGS > ATXN3-70Q 87 24.3 0.97 9.3E-01 Nurf-38 Nurf-38 {v3200} / elavGS> ATXN3-70Q 60 24.4 0.97 -9.8E-03 Hdac3 Hdac3 {v20814} / elavGS> ATXN3-70Q 90 24.4 0.97 -2.4E-02 4406-2} UAS-4406-2} / elavGS> ATXN3-70Q 86 24.6 0.98 -5.1E-01 CG4 751 CG4751 {v45530} / elavGS> ATXN3-70Q 92 24.6 0.98 -2.2E-02 CG5567 CG55 67 {v44319} / elavGS> ATXN3-70Q 72 24.6 0.98 -6.3E-03 CG15835 CG15835 {v32652} / elavGS> ATXN3-70Q 89 24.7 0.99 -7.7E-01 eRF] eRFI {v45027} / elavGS> ATXN3-70Q 99 24.7 0.99 -4.8E-01 Bap170 Bap170 {v34581} / elavGS> ATXN3-70Q 88 24.8 0.99 -4.5E-01 Sgfl] Sgfl 1 {v17166} / elavGS> ATXN3-70Q 93 24.8 0.99 -1.8E-02 rpt4 rpt4 {v49574} / elavGS> ATXN3-70Q 89 24.8 0.99 -3.0E-01 Artl Artl {v40388 } / elavGS> ATXN3-70Q 85 24.8 0.99 -6.9E-01 CG32016 CG32016 {v34755} / elavGS> ATXN3-70Q 87 24.9 0.99 -5.5E-01 mbfl mbfl {v12752} / elavGS> ATXN3-70Q 90 24.9 1.00 6.2E-01 ben ben {v9413} / elavGS> ATXN3-70Q 92 25.0 1.00 4.9E-07 Su (z) 12 Su (z) 12 {v42423} / elavGS> ATXN3-70Q 86 25.1 1.00 -4.6E-01 Hsc70-4 Hsc70-4 {v26465} / elavGS> ATXN3-70Q 89 25.1 1,00 -4,6E-01 pr-set7 pr-set7 {v34589} / elavGS> ATXN3-70Q 96 25.2 1,00 -9,6E-02 CG8417 CG8417 {v49509} / elavGS> ATXN3-70Q 94 25.2 1,00 3,3E- 01 TafS Taf5 {v45957} / elavGS> ATXN3-70Q 87 25.2 1,00 -2,1E-01 CG40351 CG4035 1 {v45267} / elavGS> ATXN3-70Q 86 25.2 1.01 -1.2E-01 YL-1 YL-1 (v21903) / elavGS> ATXN3-70Q 92 25.2 1 , 01 6,2E-01 Psc Psc {v30586) / elavGS> ATXN3-70Q 83 25.3 1.01 -1.5E-01 Snr1 Snrl {v12644} / elavGS> ATXN3-70Q 83 25.3 1.01 - 4.8E-01 Se t2 Set2 {v30707} / elavGS> ATXN3-70Q 90 25.4 1.01 -4.6E-01 Usp7 Usp7 {v18231} / elavGS> ATXN3-70Q 95 25.4 1.01 5, 0E-02 brm brm {v37721) / elavGS> ATXN3-70Q 94 25.4 1.01 2.1E-01 osa osa {v7810} / elavGS> ATXN3-70Q 87 25.4 1.01 -4.7E-01 RpL7A RpL7A {v43760} / elavGS> ATXN3-70Q 80 25.5 1.02 4.5E-01 Su (var) 3-9 Su (var) 3-9 {v39378} / elavGS> ATXN3-70Q 93 25.5 1.02 5.2 E-01 Bap55 Bap55 {v24704} / elavGS> ATXN3-70Q 84 25.6 1.02 -4.0E-01 Atac1 Atacl {v36092} / elavGS> ATXN3-70Q 25 25.6 1.02 1.5E-01 deltaCOP deltaCOP {v4I549} / elavGS> ATXN3-70Q 83 25.6 1.02 1.9E-02 Art4 Art4 {v15645} / elavGS> ATXN3-70Q 85 25.6 1.02 -2.8E -01 esc esc (v5690) / elavGS> ATXN3-70Q 89 25.6 1.02 8.8E-01 Sce Sce {v27465} / elavGS> ATXN3-70Q 57 25.6 1.02 7.2 E-01 Rpd3 Rpd3 {v30599} / elavGS> ATXN3-70Q 82 25.7 1.03 9.3E-01 Caf] Cafl {v26456} / elavGS> ATXN3-70Q 87 25.8 1.03 2.6E-02 Hcf Hcf {v46998} / elavGS> ATXN3-70Q 87 25.8 1.03 1.4E-01 RpS2 7A R pS27A {v34325} / elavGS> ATXN3-70Q 95 25.8 1.03 2.7E-02 Sirt4 Sirt4 {v40295} / elavGS> ATXN3-70Q 88 25.9 1.03 5.6E-02 Tip6O Tip60 {v22233} / elavGS> ATXN3-70Q 90 25.9 1.03 1.0E-02 CG8184 CG8184 {v26935} / elavGS> ATXN3-70Q 58 25.9 1.03 8.0E-01 Vha55 Vha55 {v46553} / elavGS> ATXN3 -70Q 88 26.0 1.04 1.0E-01 bur burst {v24153} / elavGS> ATXN3-70Q 86 26.1 1.04 2.1E-02 trx trx {v37715) / elavGS> ATXN3-70Q 88 26 , 1 1.04, 4E-01 CG1249 CG1249 {v31947} / e1avGS> ATXN3-70Q 98 26.2 1.05 8.3E-02 Iswi Iswi {v6208} / elavGS> ATXN3-70Q 90 26.3 1, 05 6.0E-04 CG15817 CG15817 {v41605} / elavGS> ATXN3-70Q 88 26.5 1.06 5.9E-02 lease lease {v48980} / elavGS> ATXN3-70Q 43 26.6 1.06 3.8E -01 Rpb4 Rpb4 {v23308} / elavGS> ATXN3-70Q 86 26.6 1.06 1.7E-03 E (z) E (z) {v27645} / elavGS> ATXN3-70Q 85 26.6 1.06 6 , 3E-03 atms atms {v20876} / elavGS> ATXN3-70Q 91 26.8 1.07 7.9E-03 Parg Parg (v23962) / elavGS> ATXN3-70Q 89 26.8 1.07 7.0E-02 Pros45 Pros45 {v24937) / elavGS> ATXN3-70Q 83 26.8 1.07 1,1E-01 Sir2 Sir2 {v23201} / elavGS> ATXN3-70Q 32 27.0 1.08 1.2E-02 TER94 TER94 {v24354 } / e lavGS> ATXN3-70Q 93 27.0 1.08 6.2E-02 CG4706 CG4706 {v34888} / elavGS> ATXN3-70Q 92 27.0 1.08 1.2E-02 Mes-4 Mes-4 {v10836} / elavGS> ATXN3-70Q 87 27.0 1.08 2.7E-03 Brel Brel {v15620} / elavGS> ATXN3-70Q 90 27.0 1.08 3.6E-03 wda wda {v34847} / elavGS> ATXN3- 70Q 78 27.2 1.09 1.5E-02 CG14619 CG14619 {v37929) / elavGS> ATXN3-70Q 85 27.3 1.09 7.5E-03 Hsc70-5 Hsc70-5 {v47745} / elavGS> ATXN3- 70Q 103 27,5 1,10 8,2E-03 trr trr {v10749} / elavGS> ATXN3-70Q 84 27,7 1,11 1,1E-05 Luci UAS-Luci / elavGS> ATXN3-70Q 82 27,9 1.11 1.4E-03 GFP UAS-GFP / elavGS> ATXN3-70Q 143 28.0 1.12 3.5E-10 Pcaf Pcaf {v21787} / elavGS> ATXN3-70Q 89 28.1 1.12 5, 1E-06 UbcD6 UbcD6 {v23229} / elavGS> ATXN3-70Q 81 28.6 1.14 2.4E-06 Cdk9 Cdk9 {v30449} / elavGS> ATXN3-70Q 62 29.3 1.17 3.1E-12 Acf1 ## EQU1 ## where: } / elavGS> ATXN3-70Q 86 30.3 1.21 5.5E-15 DNApoI-DNApoI-alpha50 {v43870} / elavGS> ATXN3-70Q 82 36.0 1.44 1.2E-30 alpha5O all males males / elavGS> ATXN3-70Q deciles 3-8 300 25.1 1.00 1,0E + 00 The life span of the individuals elavGS> ATXN3-70Q / DNApoI-alpha50 {v43870}, in which the expression level of the DNApoI-alpha50 gene is

reduced in vivo by RNA interference (RNAi), deviates significantly from the normal distribution of lifetimes (Figure 3). Thus, the inactivation of this gene (DNApol-alpha50) in flies expressing in the neurons the pathological Ataxin 3 is able to restore a lifespan similar to that of wild Drosophila with 44% increase in life span average (Figure 4a). Similarly, a similar lifespan restoration phenomenon was observed for SCA7 ataxia, with 28% increase in mean lifespan (Figure 4b) and for SCA1 ataxia, with 60% d increase in average life (Figure 4c). Table III below presents the results obtained in the various experiments carried out on the 3 Drosophila models of ataxies (SCA1, SCA3 and SCA7) expressing an RNAi specific for the DNApo1-alpha50 gene. With respect to the Drosophila SCA3 model, the ATXN3-70Q protein has been expressed either in neuronal cells or in glial cells. The ratings are the same as in Table II. The genotypes used for the different experiments for calculations of mean life span ratios and survival curve analysis are shown in bold. Table III: Results of the Ataxin 1, 3 and 7 toxicity-modifying gene screen on Drososhile models SCAT SCA3 and SCA7 remotely Genotype Condition N Duration of Ratio duration Life-span test LogRank medium mean DNApoI- alpha50 induced / control Neuronal model SCA3 DNApoI-alpha50 {v43870} / elavGS> ATXN3-70Q Induced 82 36.0 1.44 1,2E-30 males / elavGS> ATXN3-70Q deciles 3-8 Induced 300 25,1 1,00 1,0E + 00 DNApoI-alpha50 {v43870} / elavGS> ATXN3-70Q Induced 119 46.7 1.38 7.1E-44 UAS-EGFP {AH2} / elavGS> ATXN3-70Q Induced 182 33.8 1.00 1.0E + 00 DNApoI-alpha50 {v43870} / elavGS> ATXN3-70Q Induced 92 31.7 1.33 8,7E-17 UAS-GFP-IR male / elavGS> ATXN3-70Q Induced 90 23.8 1.00 1.0E + 00 DNApoI-alpha50 {v43870} / elavGS Induced 118 38.6 1.02 - 5.9E-01 DNApoI-alpha50 {v43870} / elavGS No 30 37.9 1.0 0 1.0E + 00 Induced Neuronal Model SCA7 DNApoI-alpha50 {v43870} / elavGS> SCA7-102Q Induced 195 32.7 1.28 9.7E-32 UAS-Luci / elavGS> SCA7-102Q Induced 184 25.5 1 , 00 1,0E + 00 Neural Model SCA1 DNApoI-alpha50 {v43870} / elavGS> UAS-SCA1-82Q Induced 277 44.9 1,60 3,4E-72 UAS-Luci / elavGS> UAS-SCA1-82Q Induced 267 28.0 1.00 1.0E + 00 Glial model SCA7 DNApoI-alpha50 {v43870} / dEaatl> SCA7-102Q No Induced 171 20.0 1.25 5.5E-44 UAS-Luci / dEaatl> SCA7-102Q Induced 103 16.0 1.00 1.0E + 00 DNApoI-alpha50 {v43870} / dEaatl> SCA7-102Q No Induced 125 12.5 1.22 1.3E-37 dump {V23131} / dEaatl> SCA7-102Q Not Induced 152 12.0 1.17 4.0E-26 GFP-IR / dEaatl> SCA7-102Q No Induced 235 9.2 0.89 -3.1E-21 UAS-Luci / dEaatl> SCA7-102Q No 216 10.3 1.00 Improvement in survival is also accompanied by an increase in the locomotor performance of SCA3 flies that are similar to the wild-type (unquantified) line. GMR-GAL4 was used to express an ATXN3T-78Q truncated pathological protein in the eye. Under these conditions, a strong degeneration of the eye was observed (Figure 5). This phenotype is suppressed in 10 GMR-GAL4, UAS-ATXN3T-78Q / UAS-DNApol-alpha50 (v43870) flies where DNApol-alpha50 is inactivated by RNA interference (Figure 5).

Finally, the results show that the protection afforded by the inhibition of primase (DNApoI-alpha50) also applies when the pathological protein ATXN7T-102Q is expressed in a subpopulation of glial cells (those in which the glutamate transporter dEaatl is expressed). Under these conditions, an increase in the service life of approximately 25% is observed (Table III and Figure 7). These results therefore demonstrate protection of the glial cells by inhibiting the replication of the DNA. 2.3) Genetic Screen of New Replication-Related Modifier Genes in the Drosophila Model of Spinocerebellar Ataxia SCA3 The previous results show that the reduction of the level of DNApol-alpha50 by the interfering RNA specific to DNApoI-alpha50 eliminates all toxic effects of 3 spinocerebellar ataxias by expansion of polyglutamine, SCA1, SCA3 and SCAT in a Drosophila model. The protection observed concerns both neurons and glial cells which are both critical players in degenerative processes. DNApoI-alpha50 is highly conserved in humans (orthologue of PRIM1) and plays a major role in DNA replication. The data obtained above suggest that, in neurodegenerative pathologies with polyglutamine amplification, reactivation of replication in post-mitotic neurons is a major factor of toxicity leading to associated deleterious phenotypes. It has therefore been investigated whether other genetic modifications affecting genes related to DNA replication or DNA replication control pathways can suppress the toxicity of the pathological ATXN3 protein. For this, females of genotype elavGS> ATXN3-70Q were crossed with males of different genotypes and the average life of the offspring analyzed. The genes studied are shown in Table IV below and the results are shown in Table V below.

Table IV: Genes analyzed Gene Gene orthologue Role (human or yeast) cdc6 Cdc6 Origin of pre-replication (pre-RC) DDB1 ddb1 Origin of pre-replication (pre-RC) dup Cdtl Origin of pre-replication (pre-RC) Huwel Huwel Origin of pre-replication (pre-RC) mcm2 MCM2 Origin of pre-replication (pre-RC) orc1 Orc1 Origin of pre-replication (pre-RC) or c2 Orc2 Origin of pre-replication (pre-RC) cdc45L Cdc45 Formation of the replication fork l (1) G0148 Cdc7 Formation of the DNApoI-alpha50 replication fork PRIM / Replication Step 1 DNApoI-delta POLD1 Replication step 2 DNApoI-epsilon POLE Replication step 2 cdk4 cdk4 CycA cycA cell cycle CycB cell cycle cycB CycE cell cycle cycE Cell cycle E2f E2f1 Cell cycle E2f2 E2f2 Cell cycle polo PLK1 Cell cycle string cdc25 Cell cycle tefu A TM ATM lok channel CHK2 ATM signaling path nbs1 ATM signaling pathway RAD rad50 50 Signaling-recruitment pathway ATM cdc2c cdk2 ATR signaling path CG32251 claspin ATR signaling channel grp CHK1 ATR signaling pathway p53 p53 ATR signaling pathway p53 p53 ATR signaling pathway p53 p53 ATR signaling pathway stg CDC25 ATR signaling pathway mus101 TOPBP1 ATR signaling-activation pathway mei-41A TR ATR signaling-activation pathway mus304A TRIP ATR signaling-activation pathway

Table V: Results of the screen of modifying genes using the Drosophila model SCA3 (after induction of the expression of the ATXN3-70Q protein in RU200 medium). The ratings are the same as for Table II above. The reference genotypes used for the different experiments for the calculations of the average life ratio and the survival curve analysis are indicated by an asterisk. Some experiments (represented in bold type) were performed on female flies for reasons of chromosomal location of the insertions. Table V Genotype N Duration of Ratio Duration Life Test LogRank average mean / control curve * GFP IR females / elavGS> ATXN3-70Q 118 29.3 1.00 1.0E + 00 mus101 [Dl] / elavGS> ATXN3- 70Q 117 26.5 0.90 -2.8E-08 mei-41 [e02381] / elavGS> ATXN3-70Q 122 28.0 0.95 -1.5E-08 * GFP IR male / elavGS> ATXN3-70Q 119 27.3 1.00 1.0E + 00 1 (1) G0148 [EY07177] / elavGS> ATXN3-70Q 117 24.2 0.89 -2.5E-16 rad50 [PPE] / elavGS> ATXN3-70Q 120 24 , 4 0.89 -3.8E-04 E2f2 [KG07098] / elavGS> ATXN3-70Q 116 24.4 0.89 -1.6E-09 E2f [EY14408] / elavGS> ATXN3-70Q 115 24.5 0, 90 -3,1E-04 Rpd3 [04556] ry [506] / elavGS> ATXN3-70Q 120 24,5 0,90 -1,5E-03 polo [DG12705] / elavGS> ATXN3-70Q 116 24.6 0, 90 -1.9E-06 CG32251 [EY11302] / elavGS> ATXN3-70Q 119 24.7 0.91 -5.4E-09 nbs [EY15506] / elavGS> ATXN3-70Q 114 25.1 0.92 -1, 8E-08 E2f2 [EY02995] / elavGS> ATXN3-70Q 120 26.1 0.95 -2.3E-05 p53 [5A-1-4] / elavGS> ATXN3-70Q 118 26.3 0.96 -1, 3E-04 ## STR2 ## EY09600] / elavGS> ATXN3-70Q 121 30.0 1.10 1.3E-05 mus304 [DI] / elavGS> ATXN3-70Q 120 32.4 1.18 6.2E-14 grp [EY00450] / elavGS> ATXN3-70Q 121 32.9 1, 20 1.4E-16 males / elavGS> ATXN3-70Q 300 26.8 0.98 -6.2E-01 * GFP IR females / elavGS> ATXN3-70Q 93 26.4 1.00 1.0E + 00 DNApoI- delta {V41028} females / elavGS> ATXN3-70Q 15 21.2 0.80 -7.0E-09 mus304 {V46012} females / elavGS> ATXN3-70Q 132 15.7 0.59 -1.9E-27 * GFP IR males / elavGS> ATXN3-70Q 119 24.7 1.00 1.0E + 00 cdk4 {V40576} / elavGS> ATXN3-70Q 120 19.5 0.79 -2.9E-20 mei 41 {V11251} / elavGS > ATXN3-70Q 119 20.6 0.83 -4.8E-12 mus101 {V31431} / elavGS> ATXN3-70Q 119 21.6 0.88 -9.4E-07 cycA {V32421} / elavGS> ATXN3-70Q 122 22.0 0.89 -2.6E-12 1 (1) 00148 {V40715} / elavGS> ATXN3-70Q 118 22.0 0.89 -8.1E-07 grp {V12680} / elavGS> ATXN3-70Q 123 22.3 0.91 -8.0E-05 tefu {V22502} / elavGS> ATXN3-70Q 90 22.6 0.92 -1.0E-04 cdc6 {V46927} / elavGS> ATXN3-70Q 31 23.4 0.95 -2.1E-01 orcl {V46522} / elavGS> ATXN3-70Q 118 24.4 0.99 3.9E-02 p53 {V45138} / elavGS> ATXN3-70Q 115 24.4 0.99 3, 0E-01 cdc45L {V41084} / elavGS> ATXN3-70Q 117 24.8 1.00 4.7E- 04 lok {V44980} / elavGS> ATXN3 -70Q 122 24.8 1.01 6.9E-01 orc2 {V47603} / elavGS> ATXN3-70Q 119 25.5 1.04 4.9E-03 p53 {V 106921 / elavGS> ATXN3-70Q 117 25.6 1.04 6.3E-06 cycB {V43772} / elavGS> ATXN3-70Q 122 25.7 1.04 4.1E-03 cycE {V52662} / elavGS> ATXN3-70Q 123 25.8 1.05 7.6E-05 DNA pol epsilon {V13647} / elavGS> ATXN3-70Q 116 25.8 1.05 1.8E-04 1 (1) G0148 {V24186} / elavGS> ATXN3-70Q 118 27.1 1.10 1.8E-12 mcm2 {V10967} / elavGS> ATXN3-70Q 30 27.3 1.17 7.5E-07 dup {V2313 1} / elavGS> ATXN3-70Q 117 27.7 1, 12 2,3E-19 * males / elavGS> ATXN3-70Q deciles 3-8 300 24,4 0,99 2,3E-01 * UAS-GFP-IR female / elavGS> ATXN3-70Q 110 22,8 1,00 1.0E + 00 1 (1) G0148 [EY07177] females NV / elavGS> ATXN3-70Q 25 22.1 0.97 -1.7E-04 mei-411e02381] females / elavGS> ATXN3-70Q 13 22.4 0 , 98 -1,6E-01 UAS-luci female / elavGS> ATXN3-70Q 20 23,3 1,02 -6,5E-02 1 (1) G0148 [EY07177] females / elavGS> ATXN3-70Q 98 24.4 1.07 -5.8E-02 Huwel {EYOI689} females / elavGS> ATXN3-70Q 31 36.3 1.59 2.6E-20 * UAS-GFP-IR male / elavGS> ATXN3-70Q 90 23.8 1 , 00 1.0E + 00 cdc6 {v4 6927} / elavGS> ATXN3-70Q 29 15.7 0.66 -8.6E-13 UAS-S6K / elavGS> ATXN3-70Q 31 16.1 0.68 -1.7E-13 cdk4 {v40576} / elavGS> ATXN3-70Q 80 17.6 0.74 -2.0E-08 cdc2c [2] / elavGS> ATXN3-70Q 58 21.1 0.89 2.1E-01 mei-41 {v11251} / elavGS> ATXN3-70Q 119 21.9 0.92 -4.3E-02 E2F2 {KG07098} / elavGS> ATXN3-70Q 60 22.1 0.93 -5.6E-01 CG32251 [EY11302] / elavGS> ATXN3-70Q 111 22.3 0.94 -5.6E-02 UAS-luci male / elavGS> ATXN3-70Q 52 22.4 0.94 8.9E-02 mus 101 {v31431} / elavGS> ATXN3-70Q 120 22.4 0.94 - 2,4E-02 cycA {v32421} / elavGS> ATXN3-70Q 57 22.8 0.96 -7.2E-01 mei-41 [e02381] males (controls) / elavGS> ATXN3-70Q 37 22.9 0, 96 -1.7E-02 1 (1) G0148 {v40715} / elavGS> ATXN3-70Q 120 23.0 0.97 -7.1E-01 UAS-cycE / elavGS> ATXN3-70Q 81 23.1 0.97 3.6E-02 UAS-E2F void p 1 717 / elavGS> ATXN3-70Q 90 23.2 0.98 8.1E-01 UAS-cyceE p1396 / elavGS> ATXN3-70Q 65 23.3 0.98 -1, 2E-02 dup {v23131} / elavGS> ATXN3-70Q 88 24.0 1.01 7.7E-01 grp [DG25208] / elavGS> ATXN3-70Q 51 24.2 1.02 -6.3E-02 UAS- cycA / elavGS> ATXN3-70Q 48 24.8 1.04 5.9E-01 mus 101 [D 1] / elavGS> ATXN3-70Q 123 24.8 1.04 5.5E-02 grp {v12680} / elavGS> ATXN3-70Q 47 25.4 1.07 2.9E-02 nbs [EY15506] / elavGS> ATXN3-70Q 116 25.4 1.07 8.2E -02 w / elavGS> ATXN3-70Q 116 25.8 1.09 1.4E-03 DDB 1 [EY01408] / elavGS> ATXN3-70Q 55 25.9 1.09 9.4E-03 grp [06034] / elavGS > ATXN3-70Q 80 26.3 1.13 3.0E-02 UAS-string / elavGS> ATXN3-70Q 79 26.8 1.13 1.4E-06 DNApo1-alpha50 {v43870} / elavGS> ATXN3-70Q 92 31.7 1.33 8.7E-17 grp [EY00450] / elavGS> ATXN3-70Q 116 32.8 1.38 1.3E-27 * males / elavGS> ATXN3-70Q deciles 3-8 300 23.6 These Genetic screens have identified several other genes involved in DNA replication that may modulate SCA3 pathology. These are the mcm2 and dup / Cdtl genes required for replication and whose inactivation saves the pathological phenotype, as well as the Huwel gene, a replication inhibitor whose gain of function has a protective effect in SCA3 pathology. . On the other hand, the gain in function of the grp / Chkl kinase, a major effector of the ATR pathway involved in the control of DNA replication, also saves the pathological phenotype. Thus, several genes that play a role in the process of DNA replication and its control appear as potential therapeutic targets.

2.4) Pharmacological tests A pharmacological test with hydroxyurea (antireplicative compound) was performed on the neuronal Drosophila model SCA3. The results are shown in Table VI below.

Table VI: Results of pharmacological tests with hydroxyurea. Hydroxyurea was incorporated into the nutrient medium at the concentration reported in column 4. RU486 was also incorporated into this medium at 200 μg / ml. The genotype of the tested males is reported in column 2. Table VI Genotype Concentration N Duration of Ratio Lifespan Test of (mg / ml) mean life LogRank medium individuals treated with hydroxyurea / untreated elavGS> ATXN3-70Q-WT2 / 0 88 5,6 1,00 1,0E + 00 elavGS> ATXN3-70Q-WT2 elavGS> ATXN3-70Q-WT2 / 0,70 86 7,6 1,34 8,2E-05 elavGS> ATXN3-70Q- WT2 elavGS> ATXN3-70Q-WT2 / 2.1 82 7.2 1.27 2.5E-03 elavGS> ATXN3-70Q-WT2 elavGS> ATXN3-70Q-WT2 / 5.6 88 5.4 0,96 - 7.1E-01 elavGS> ATXN3-70Q-WT2 elavGS> ATXN3-70Q-WT2 / 129 18.0 1.00 1.0E + 00 ATXN3-70Q-WT3 elavGS> ATXN3-70Q-WT2 / 0.14 133 18 , 0 1,00 6,1E-01 ATXN3-70Q-WT3 elavGS> ATXN3-70Q-WT2 / 0,35 136 18,4 1,02 2,4E-01 ATXN3-70Q-WT3 elavGS> ATXN3-70Q-WT2 / 0.70 136 18.6 1.03 4.9E-01 ATXN3 -70Q-WT3 A positive effect was obtained with hydroxyurea at a concentration of 0.7 mg / ml on homozygous males elavGS> ATXN3-70QWT2 / elavGS> ATXN3-70Q-WT2. However, this effect was not observed in heterozygous males of the elavGS> ATXN3-70Q-WT2 / ATXN3-70Q-WT3 genotype with longer longevity (the WT2 and WT3 lines correspond to independent transgenic UAS-ATXN3-70Q transgene).

REFERENCES Andorfer, C., et al. (2005). J Neurosci 25, 5446-5454. Arezi, B., and Kuchta, R.D. (2000). Trends Biochem Sci 25, 572-576. Bilen, J., and Bonini, N.M. (2005). Annu Rev Genet 39, 153-171.

Bonini, N.M., and Fortini, M.E. (2003). Annu Rev Neurosci 26, 627-656. Brand AH, and Perrimon N (1993) Development 118, 401- 415. Cimprich, K.A., and Cortez, D. (2008). Nat Rev Mol Cell Biot 9, 616-627. Copani, A., et al. (2002). Faseb J 16, 2006-2008. Copani, A., et al. (2006). J Neurosci 26, 10949-10957.

Copani, A., et al. (2007). Biochim Biophys Acta 1772, 409-412. Copani, A., et al. (2008). Curr Med Chem 15, 2420-2432. Fernando-Funez, P., et al., (2000). Nature 408, 101-106. Gatchel, J.R., and Zoghbi, H.Y. (2005). Nat Rev Genet 6, 743-755. Herrup, K., et al. (2004). J Neurosci 24, 9232-9239.

Khurana, V., et al. (2006). Curr Biol 16, 230-241. Khurana, V., and Feany, M. B. (2007). Biochim Biophys Acta 1772, 446-456. Lao-Sirieix, S.H., et al. (2005). Trends Genet 21, 568-572. Latouche, M., et al. (2007). J Neurosci 27, 2483-2492. Lee, S.S., et al. (2003). Neurobiol Aging 24, 687-696.

Mueller, T., et al. (2009). Human Mol Gen 18, 3334-3343. Muzi-Falconi, M., et al. (2003). ScientificWorldJournal 3, 21-33. Peto, R. and Peto, J. (1972). Journal of the Royal Statistical Society; Series A; 135, 185-206. Ranganathan, S., and Bowser, R. (2003). Am J Pathol 162, 823-835.

Rival, T., et al. (2004). Curr Biot 14, 599-605. Roignant, J.Y., et al., (2003). RNA 9, 299-308. Blood, T.K., and Jackson, G.R., (2005). NeuroRx 2, 438-446. Sclafani, R.A., and Holzen, T.M. (2007). Annu Rev Genet 41, 237-280. Shibutani S.T., et al. (2008). Dev Cell 15, 890-900.

Shulman, J.M., et al. (2003). Curr Opin Neurol 16, 443-449. Soong, B.W., and Paulson, H.L. (2007). Curr Opin Neuro120, 438-446.

Ueberham, U., and Arendt, T. (2005). Curr Drug Targets CNS Neurol Disord 4, 293-306. Warrick, J.M., et al. (1998). Celi 93, 939-949. Zoghbi, H.Y., and Botas, J. (2002). Trends Genet 18, 463-471.5

Claims (8)

REVENDICATIONS1. Use of a DNA replication inhibitor for the preparation of a pharmaceutical composition for the treatment of a neurodegenerative disease by polyglutamine expansion. 2. Use according to claim 1, characterized in that said inhibitor of DNA replication is an inhibitor of a protein selected from the factors of initiation of replication, helicases, primases, DNA polymerases, DNA ligases, topoisomerases, nuclear antigens of nuclear proliferation (PCNA), replication factors A and C. 3. Use according to claim 1 or claim 2, characterized in that said inhibitor of DNA replication inhibits the expression of a gene selected from priml, mcm2 and Cdtl genes and the orthologous genes thereof . 4. Use according to claim 1 or claim 2, characterized in that said inhibitor of DNA replication activates or increases the expression of a gene selected from the Huwel and Chkl genes and the orthologous genes thereof . 5. Use according to any one of claims 1 to 4, characterized in that said inhibitor of DNA replication is selected from an antibody, a nucleic acid, a peptide, a protein. 6. Use according to claim 1, characterized in that said inhibitor of the replication of the DNA is hydroxyurea. 7. Use according to any one of claims 1 to 6, characterized in that said neurodegenerative disease by expansion of polyglutamine is selected from Huntington's disease, autosomal dominant spinocerebellar ataxias 1, 2, 3, 6, 7 and 17, dentatorubro-pallido-luysian atrophy and Kennedy's disease. 8. Use according to claim 7, characterized in that said neurodegenerative disease by expansion of polyglutamine is selected from autosomal dominant spinocerebellar ataxias 1, 2, 3, 6, 7 and 17.