WO2011042872A1

WO2011042872A1 - Use of a dna replication inhibitor for treating neurodegenerative diseases caused by polyglutamine expansion

Info

Publication number: WO2011042872A1
Application number: PCT/IB2010/054522
Authority: WO
Inventors: Hervé Tricoire; Véronique Monnier; Anita Reinhardt; Michael Rera
Original assignee: Universite Paris Diderot - Paris 7
Priority date: 2009-10-08
Filing date: 2010-10-06
Publication date: 2011-04-14
Also published as: FR2951083A1; WO2011042872A4

Abstract

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

Description

USE OF A DNA REPLICATION INHIBITOR FOR THE TREATMENT OF NEURODEGENERATIVE DISEASES BY EXPANSION

POLYGLUTAMINE

The present invention relates to the field of medicaments for the treatment of neurodegenerative diseases by polyglutamine expansion, more particularly 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 markers of the cell cycle, such as α, δ, β, and primase DNA polymerases, and replicate their DNA (Herrup et al., 2004, Ranganathan and Bowser, Lee et al., 2003; 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 peptide Αβ42, that the toxicity of this peptide is reduced in cells where the DNA polymerase beta is inactivated (Copani et al, 2002, 2006, 2007 and 2008).

In contrast, with respect to neurodegenerative diseases by polyglutamine expansion, including dominant spinocerebellar ataxias, Khurana et al (2006) have shown using a Drosophila model of tauopathy that the expression of the MJD- 78Q (MJD protein, also called ATXN3, having an expansion of 78 glutamines) induced apoptotic neurodegeneration, without the authors being able to observe 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 Hsp90 ("chaperone" protein, which plays a role in protecting the three-dimensional folding of proteins), such as derivatives of geldanamycin. To the knowledge of the inventors, no data concerning a possible influence of deacetylases on the transcription of genes related to DNA replication has been documented in post-mitotic cells such as neurons.

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 polyglutaminic 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 allows 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 were then used to identify, 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 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 DNApol-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 SCA7 studied. The observed cell protection concerns both neurons and glial cells. Rescue of neurodegeneration in the eye has also been observed by the inventors on a Drosophila model of Huntington's disease in which the level of the DNApol-alpha50 protein is reduced by RNA interference.

Other genetic screens on the Drosophila SCA3 model have enabled the inventors to identify several other actors of DNA replication capable of modulating the SCA3 pathology: these are the mcm2, dup / Cdtl, DNApol-alpha73IPOLA2 and timeoutlTimeless necessary for the replication of the DNA and whose inactivation saves the pathological phenotype, the gene Huwel, a DNA replication inhibitor, whose gain of function has a protective effect in the pathology SCA3, as well as the gene grp / Chkl, coding for a major kinase of the ATR pathway ("Ataxia Telangiectasia and RadS-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 the cell cycle that precedes the later phases of the cycle (G2, M, Gl) (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 required 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), and TIM protein ("Timeless") (Benna et al, 2010);

proteins controlling the course of the cell cycle, such as cdk kinases and cyclins (Sclafani and Holzen, 2007);

DNA polymerases such as the subunit B of DNA polymerase alpha (POLA2) (Takahashi et al., 1996).

The subject of the present invention is therefore the use of an inhibitor of DNA replication, which DNA replication inhibitor is an inhibitor of a protein selected from primases, replication, helicases, DNA polymerases, DNA ligases, topoisomerases, kinases, nuclear proliferation nuclear antigens (PCNA), replication factors A and C, preferably a primase, 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 the tissues, preferably the neuronal and / or glial cells, of said subject compared to a level of replication of the measured DNA in the absence of the administration. 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 ³ H-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 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.

The DNA replication inhibitor can be a chemical or biological compound of natural or synthetic origin. It can 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 the replication of the DNA of said compound must be preponderant at the dose (or concentration) used compared to its other actions.

According to a preferred embodiment of the invention, said inhibitor of DNA replication is an inhibitor of DNA replication of post-mitotic cells, such as neurons.

According to another preferred embodiment of the invention, said inhibitor of DNA replication inhibits the expression of a gene selected from prim1 genes (coding for primase DNA), mcm2 (coding for protein "minichromosome maintenance complex component 2"), Cdtl (coding for the "chromatin licensing and DNA replication factor 1" protein), PLOA2 (encoding the DNA polymerase alpha subunit B), Timeless (encoding the protein TIM) and the orthologous genes of these genes, preferably the prim1 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 HECT protein, UBA and WWE containing domain 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 (e.g., cyclophosphamide, platinum salts), an intercalating agent (e.g., anthracyclines) or a nucleic acid analogue (e.g., 5-FU).

By subject is meant a mammal, preferably a human.

According to another preferred embodiment of the invention, said neurodegenerative polyglutamine expansion disorder is selected from Huntington's disease, autosomal dominant spinocerebellar ataxias 1, 2, 3, 6, 7 and 17, dentato-rubro atrophy. -pallido-luysian and Kennedy's disease (or spino-bulbar muscular atrophy), preferably autosomal dominant spinocerebellar ataxias and Huntington's disease, 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 the skilled person. 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 DNA replication inhibitors do not necessarily cure the subject with the disease, but may 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 become apparent from the experimental examples below, as well as to the accompanying drawings, in which:

FIG. 1 represents the principle of establishing a Drosophila model 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;

FIG. 2 represents the principle of a genetic screen by interfering RNA inactivation. Drosophila expressing a pathological protein (eg, Ataxin 3) in a specific tissue (eg, neurons) are crossed with individuals carrying an inverted tandem repeat of a portion of the cDNA of a gene modifying interest (rated 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 RNA interference, 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 / DNApol-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 control individuals 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; - Figure 5: 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 picture) and to the phenotype restored by decreasing the expression of primase induced by interfering RNA (photo right);

- 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 TATXN3-70Q is not modified by the presence of the transposon DNApol-a50- {v43870}. Genotypes: elav-GAL4GS, UAS-ATXN3- 70Q / UAS-EGFP (left track); elav-GAL4GS, UAS-ATXN3-70Q / DNApol-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. Simultaneous reduction of the level of primase (DNApolalpha50) (solid triangles) improves survival in this glial model of ataxia;

FIG. 8: photographs showing, in Drosophila, the neurodegeneration induced by the expression in the eye of the truncated pathological human Huntingtin protein (photo A) compared to the phenotype restored by the decrease in the expression of primase induced by Interfering RNA (photo B).

EXAMPLES 1: IDENTIFICATION OF GENES INVOLVED IN DNA REPLICATION LIKELY TO MODULATE DOMINANT SPINOCEREBELLAR ATAXIA SCA1, SCA3 AND SCA7

1) Materials and Methods

Drosophila strains and culture

Three neuronal models of different spinocerebellar ataxias (SCA1, SCA3 and SCA7) were generated by combining a pilot line expressing the GAL4 protein under the control of the specific promoter of the differentiated neurons elavGS (Latouche et al, 2007) and lines containing a transposon (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 interference-specific RNA of a modifier 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 interfering RNA inactivation.

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, 1.1% 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

Female 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) whose expression does not 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 (RU0). 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. 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 DNApol-alpha50 {v43870} and UAS-GFP-IR were cultured 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 Mot and Immunodetection of Ataxin 3

The expression of Ataxin 3 in Drosophila genotype elavGS> ATXN3-70Q / UAS-EGFP and elavGS> ATXN3-70Q / DNApol-alpha50 {v43870} grown for 4 days on inducible medium was analyzed by Western blot. 10 flies of each condition (induction or not of the expression of the pathological protein) were crushed in ΙΟΟμί 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) 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.

2) Results

2.1) Characterization of Drosophila models of spinocerebellar ataxia SCA1, SCA3 and SCA 7.

Drosophila models of spinocerebellar ataxia type SCA1, SCA3 and SCA7 have been generated and characterized by various studies: phenotype analysis 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 35% decrease in the longevity of individuals expressing pathological Ataxin 3 compared with individuals similarly uninduced genotype (with RU486), not expressing 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, the genotype of which is indicated in column 1, were placed under induced conditions (the flies are placed on medium containing 200 of RU486, medium RU200) 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 the pathological Ataxin 3

GFP IR / elavGS> ATXN3-70Q No Induced 27 44.1

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 SCA 7) by Primase Inactivation

98 lines expressing an interfering RNA, 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 MRA. For this, females of the genotype elavGS UAS-ATXN1-82Q, elavGS UAS-ATXN3-70Q and elavGS UAS-ATXN7T-102 Q were respectively crossed with males of different genotypes and the offspring was analyzed (according to the principle of genetic screen shown in 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 DNApol-alpha50 is inactivated (second last line of the table). Table II

Gene Genotype N Duration of Ratio Life Test Duration of LogRank Mean Life after Average Elimination

/ curve of the 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

## EQU1 ## v28982} / elavGS> ATXN3-70Q 99 21.5 0.86 -3.7E-11

Rpn6 pn6 {vl 8022} / elavGS> ATXN3-70Q 96 21.5 0.86 -2.9E-22

CGI 4224 CG14224 {v47447} / elavGS> ATXN3-70Q 92 21.7 0.86 -2.5E-09

CG2051 CG2051 {v33459} / 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} / elavGS> ATXN3 -70Q 100 21, 8 0.87 -2.2E-09 1 (l) G0148 1 (1) G0148 (v24184) / elavGS> ATXN3-70Q 74 22.0 0.88 -2.4E-01 not 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 cn {vl 1322} / 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

## EQU1 ##

CG4165 CG4165 {v41976} / elavGS> ATXN3-70Q 93 23.2 0.93 -2.7E-06

CG14712 CG 14712 (v 18347) / elavGS> ATXN3-70Q 98 23.2 0.93 -3.2E-03

Case Case {vl2648} / elavGS> ATXN3-70Q 96 23.3 0.93 -l, 9E-03

SmD3 SmD3 {v35934} / elavGS> ATXN3-70Q 85 23.5 0.94 -L, 3E-03

CG6905 CG6905 {vl3492} / elavGS> ATXN3-70Q 92 23.5 0.94 -I, 0E-02

Usp36 Usp36 {vl 1 152} / elavGS> ATXN3-70Q 91 23.6 0.94 -6.4E-04

Tafl Tafl {v41099} / elavGS> ATXN3-70Q 90 23.6 0.94 -Ι, ΟΕ-03

HDAC4 HDAC4 {v20522} / elavGS> ATXN3-70Q 89 23.7 0.95 -2.6E-05 cdk7 cdk7 {vl0442} / 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 Pp 1 alpha-96 A {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

PICKl PICKl {v22269} / elavGS> ATXN3-70Q 91 24.1 0.96 -2.5E-01 Mid-2 Mi-2 {vl0766} / elavGS> ATXN3-70Q 90 24.1 0.96 -5.5E-03

SirO Sirt2 {v21999} / elavGS> ATXN3-70Q 86 24.1 0.96 -9, 1E-03 enc encl5291} / elavGS> ATXN3-70Q 85 24.2 0.97 -6.0E-02

4406-1-1M} UAS-4406- 1 - 1 M} / 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

CG4751 CG4751 {v45530} / elavGS> ATXN3-70Q 92 24.6 0.98 -2.2E-02

CG5567 CG5567 {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 eRFl eRFl {v45027} / elavGS> ATXN3-70Q 99 24.7 0.99 -4.8E-01

ΒαρΠΟ Bap 170 {v34581} / elavGS> ATXN3-70Q 88 24.8 0.99 -4.5E-01 gfll Sgfl 1 {vl7166} / elavGS> ATXN3-70Q 93 24.8 0.99 -L, 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

### 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

Taf5 Taf5 {v45957} / elavGS> ATXN3-70Q 87 25.2 1,00 -2,1E-01

CG40351 CG40351 {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

Snrl Snrl {vl2644} / elavGS> ATXN3-70Q 83 25.3 1.01 -4.8E-01

Set2 Set2 {v30707} / elavGS> ATXN3-70Q 90 25.4 1.01 -4.6E-01

Usp7 Usp7 {vl 8231} / 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 RpL7 To {v43760} / elavGS> ATXN3 -70Q 80 25.5 1.02 4.5E-01

Sn (var) 3-9 Su (var) 3-9 {v39378} / elavGS> ATXN3-70Q 93 25.5 1, 02 5.2E-01

Bap55 Bap55 {v24704} / elavGS> ATXN3-70Q 84 25.6 1, 02 -4.0E-01

Atacl Atacl {v36092} / elavGS> ATXN3-70Q 25 25.6 1, 02 1, 5E-01 deltaCOP deltaCOP {v41549} / elavGS> ATXN3 -70Q 83 25.6 1, 02 l, 9E-02

Art4 Art4 {vl 5645} / 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 7.2E-01

Rpd3 Rpd3 {v30599} / elavGS> ATXN3-70Q 82 25.7 1, 03 9.3E-01 Cafl 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

RpS27A RpS27A {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

Tip60 Tip60 {v22233} / elavGS> ATXN3-70Q 90 25.9 1.03 l, 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 br bur {v24153} / elavGS> ATXN3-70Q 86 26.1 1.04 2, lE-02 trx trx {v37715 } / elavGS> ATXN3-70Q 88 26.1 1.04 3.3E-01

CG1249 CG1249 {v31947} / elavGS> 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 l, 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 l, 2E-02

TER94 TER94 {v24354} / elavGS> ATXN3-70Q 93 27.0 1.08 6.2E-02

CG4706 CG4706 {v34888} / elavGS> ATXN3-70Q 92 27.0 1.08 l, 2E-02

Mes-4 Mes-4 {vl0836} / elavGS> ATXN3-70Q 87 27.0 1.08 2.7E-03

Brel Brel {vl5620} / elavGS> ATXN3-70Q 90 27.0 1.08 3.6E-03 wda wda {v34847} / elavGS> ATXN3 -70Q 78 27.2 1.09 l, 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 {vl 0749} / elavGS> ATXN3-70Q 84 27.7 1.11 Ι, ΙΕ- 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, lE-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

Acfl Acfl {v33447} / elavGS> ATXN3-70Q 13 29.5 1,18 3,3E-02

CG17293 CG17293 {v25248} / elavGS> ATXN3-70Q 86 29.8 1.19 7.3E-13

CG7597 CG7597 {v25508} / elavGS> ATXN3-70Q 86 30.3 1, 21 5.5E-15

DNApol- DN Apol-alpha50 {v43870} / elavGS> ATXN3 -70Q 82 36.0 1, 44 l, 2E-30 alpha50

all male males / elavGS> ATXN3-70Q deciles 3-8 300 25.1 1, 00 l, 0E + 00 The lifespan of elavGS> ATXN3-70Q / DNApol-alpha50 {v43870} individuals, in which the expression level of the DNApol-alpha50 gene is reduced in vivo by RNA interference (RNAi), deviates significantly from the normal distribution. 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 DNApol-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 the calculations of the average life ratio and the analysis of the survival curves are indicated in bold type.

Table III: Screening Results of the Ataxin 1, 3 and 7 Toxicity Modifying Genetic Screens on Drosophila Models SCA1, SCA3 and SCA7 respectively

Genotype Condition N Duration of Ratio Duration Average Life Test LogRank average

DNApol-alpha50

induced /

control

SCA3 neuronal model

DNApol-alpha50 {v43870} / elavGS> ATXN3-70Q Induced 82 36.0 1.44 l, 2E-30 maize garlic / elavGS> ATXN3-70Q deciles 3-8 Induced 300 25,1 1,00 l, 0E + 00

DNApol-alpha50 {v43870} / elavGS> ATXN3-70Q Induced 1 19 46.7 1.38 7, l E-44

UAS-EGFP {AH2} / elavGS> ATXN3-70Q Induced 182 33.8 1.00 l, 0E + 00

DNApol-alpha50 {v43870} / elavGS> ATXN3-70Q Induced 92 31.7 1.33 8.7E-17

UAS-GFP-IR maie / elavGS> ATXN3-70Q Induced 90 23.8 1.00 l, 0E + 00

DNApol-alpha50 {v43870} / elavGS Induced 1 18 38.6 1, 02 -5.9E-01

DNApol-aIpha50 {v43870} / elavGS No 30 37.9 1,00 l, 0E + 00 armature

Neural model SCA7

DN Apol-alpha50 {v43870} / elavGS> SC A7- 102Q Induced 195 32.7 1.28 9.7E-32

UAS-Luci / elavGS> SCA7-102Q Induced 184 25.5 1.00 l, 0E + 00

SCAl neuronal model

DNApol-alpha50 {v43870} / elavGS> UAS-SCAl-82Q Induced 277 44.9 1.60 3.4E-72

UAS-Luci elavGS> UAS-SCAl-82Q Induced 267 28,0 1,00 l, 0E + O0

SCA7 glial model

DNApol-alpha50 {v43870} / dEaatl> SCA7-102Q Not Induced 171 20.0 1.25 5.5E-44

No

UAS-Luci / dEaatl> SCA7-102Q 103 16.0 1.00 l, 0E + 00

armature

DNApol-alpha50 {v43870} / dEaat 1> SC A7- 102Q No Induced 125 12.5 1.22 l, 3E-37 dup {V23131} / dEaatl> SCA7-102Q No Induced 152 12.0 1, 17 4.0E -26

GFP-IR / dEaatl> SCA7-102Q No Induced 235 9.2 0.89 -3.1E-21

No

UAS-Luci / dEaatl> SCA7-102Q 216 10.3 1.00 l, 0E + 00

armature

The improved survival is also accompanied by an increase in locomotor performance of SCA3 flies that are similar to the wild (unquantified) line.

GMR-GAL4 was used to express a truncated ATXN3T-78Q pathological protein in the eye. Under these conditions, a strong degeneration of the eye has been observed (Figure 5). This phenotype is suppressed in the 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 primase inhibition (DNApol-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 glial cells by inhibition of DNA replication.

2.3) Genetic Screening 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 DNApol-alpha50-specific interfering RNA makes it possible to eliminate all the toxic effects of 3 spinocerebellar ataxias by polyglutamine, SCA1, SCA3 and SCA7 expansion in a Drosophila model. Protection observed involves both neurons and glial cells which are both critical players in degenerative processes.

DNApol-alpha50 is very strongly conserved in humans (orthologue of PRIM1) and plays a major role in the replication of DNA.

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 ddbl 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) orcl Orcl Origin of pre-replication (pre-RC) orc2 Orc2 Origin of pre-replication (pre-RC) cdc45L Cdc45 Formation of the replication fork l (l) G0148 Cdc7 Formation of the replication fork

DNApol-alpha50 priml Replication Step 1

DNApol-delta POLD1 Replication Step 2

DNApol-epsilon POLE Replication Step 2

cdk4 cdk4 Cell Cycle

cycA cycA Cell cycle

cycB cycB Cell cycle

cycE cycE Cell cycle

E2f E2fl Cell cycle

E2f2 E2J2 Cell cycle

polo PLK1 Cell cycle

string cdc25 Cell cycle tefu ATM ATM lok signaling channel CHK2 ATM nbs1 signaling channel NBS1 ATM50 signaling-recruiting path RAD50 ATM signaling-recruiting pathway cdc2c cdk2 ATR signaling pathway

CG32251 claspin ATR signaling channel

grp CHK1 ATR p53 p53 signaling pathway ATR p53 p53 signaling pathway ATR p53 p53 signaling pathway ATR stg CDC25 signaling pathway ATR muslOl signaling pathway TOPBP1 ATR signaling-activation pathway mei-41 ATR ATR signaling-activation channel 304 ATRIP ATR signaling-activation pathway

DNApol-alpha 73 POLA2 Replication Step 1

Timeout Timeless (TIM) Cell Cycle

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 Ratio Duration Duration LogRank Average Life Lifespan Test /

curve

witness

* GFP IR females / elavGS> ATXN3-70Q 118 29.3 1.00 l, 0E + 00 muslOl [Dl] / elavGS> ATXN3-70Q 117 26.5 0.90 -2.8E-08 mei-41 [e02381 ] / elavGS> ATXN3-70Q 122 28.0 0.95 -L, 5E-08

* GFP IR male / elavGS> ATXN3-70Q 119 27.3 1.00 Ι, ΟΕ + 00

1 (1) G0148 [EY07177] / elavGS> ATXN3-70Q 117 24.2 0.89 -2.5E-16 rad50 [EP1] / elavGS> ATXN3-70Q 120 24.4 0.89 -3.8E-04

E2f2 [KG07098] / elavGS> ATXN3-70Q 116 24.4 0.89 -L, 6E-09

E2f [EY14408] / elavGS> ATXN3-70Q 115 24.5 0.90 -3, IE-04 Rpd3 [04556] ry [506] / elavGS> ATXN3-70Q 120 24.5 0.90 -l, 5E-03 polo [DG 12705] / 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 [EY 15506] / elavGS> ATXN3-70Q 114 25.1 0.92 -L, 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 -l, 3E- ## STR1 ## ## EQU1 ## 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 l, 0E + 00

DNApol-delta {V41028} females / eIavGS> ATXN3-70Q 15 21.2 0.80 -7.0E-09 mus304 {V46012} females / elavGS> ATXN3-70Q 132 15.7 0.59 -l, 9E-27

* GFP IR males / elavGS> ATXN3-70Q 119 24.7 1.00 l, 0E + 00 cdk4 {V40576} / elavGS> ATXN3-70Q 120 19.5 0.79 -2.9E-20 mei 41 {VI 1251 } / elavGS> ATXN3-70Q 119 20.6 0.83 -4.8E-12 muslOl {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) GO 148 (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 tefii {V22502} / elavGS> ATXN3-70Q 90 22.6 0.92 -l, 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 4JE-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, 1E-03 p53 {V10692} / elavGS> ATXN3-70Q 117 25.6 1.04 6.3E-06 cycB {V43772} / elavGS> ATXN3-70Q 122 25.7 1.04 4, lE-03 cycE {V52662} / elavGS> ATXN3-70Q 123 25.8 1.05 7.6E-05

## STR1 ## mcm2 {V10967} / elavGS> ATXN3-70Q 30 27.3 1.17 7.5E-07 dup {V23131} / 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 l, 0E + 00

1 (1) G0148 [EY07177] females NV / elavGS> ATXN3-70Q 25 22.1 0.97 -l, 7E-04 mei-41 [e02381] 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 {EY01689} females / elavGS> ATXN3-70Q 31 36.3 1.59 2,6E-20

* UAS-GFP-IR male / elavGS> ATXN3-70Q 90 23.8 1.00 l, 0E + 00 cdc6 {v46927} / elavGS> ATXN3-70Q 29 15.7 0.66 -8.6E-13

UAS-S6K / elavGS> ATXN3-70Q 31 16.1 0.68 -1JE-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 {vl 1251} / 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 muslOl {v31431} / elavGS> ATXN3-70Q 120 22.4 0.94 -2.4E-02 cycA {v32421} / ## EQU1 ## ) 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 pl717 / elavGS> ATXN3-70Q 90 23.2 0.98 8.1E-01

UAS-cycE _P 1396 / elavGS> ATXN3-70Q 65 23.3 0.98 -l, 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 muslOl [Dl] / elavGS> ATXN3-70Q 123 24.8 1.04 5.5E-02 grp {vl2680} / 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 l, 4E-03

DDBl [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 l, 4E-06 timeout {v22592} / elavGS> ATXN3-70Q 113 30.4 1.12 1.11-02

DNApol-alpha73 {vl08579} / elavGS> ATXN3-70Q 125 30.6 1.12 2.2E-02

DNApol-alpha73 {vl08579} / elavGS> ATXN3-70Q 121 31.6 1, 22 l, 8E-08 DNApol-alpha50 {v43870} / elavGS> ATXN3-70Q 92 31.7 1.33 8 / 7E-17 timeout [c06976] / elavGS> ATXN3-70Q 114 31.8 1.12 1.2Ε-06 timeout {v22592} / elavGS> ATXN3-70Q 149 32.6 1.25 5.7E-20 grp [EY00450] / elavGS> ATXN3-70Q 116 32.8 1.38 l, 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. These screens also made it possible to identify the genes dPOLA2 and Tequeless that are necessary for replication and whose inactivation partially restores the wild-type phenotype. Thus, several genes that play a role in the process of DNA replication and its control appear as potential therapeutic targets.

EXAMPLE 2: DEMONSTRATING THE EFFECT OF REDUCING THE EXPRESSION OF PRIMASE ON HUNTINGTON'S DISEASE IN DROOSOPHILE

1) Materials and Methods

Drosophila strains and culture

For the Huntington's disease model study, the GMR-UAS-Htt-93Q line was created from a pUAS-Htt 93Q line (Steffan et al, 2001).

The culture conditions are identical to those described in Example 1.

Analysis of the phenotype of the eye

Females of genotype GMR> Htt-93Q and males of genotype DNApol-alpha50 {v43870} and UAS-GFP-IR were raised to 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. 2) Results

GMR-GAL4 was used to express the truncated human pathogenic Huntingtin protein Htt-93Q (intron 1) in the eye. Under these conditions, a strong degeneration of the eye has been observed (FIG. 8A). This phenotype is suppressed in UAS-Htt-93Q / UAS-DNApol-alpha50 {v43870} flies where DNApol-alpha50 is inactivated by RNA interference (Figure 8B).

REFERENCES

Andorfer, C, et al. (2005). J Neurosci. 25, 5446-5454.

Arezi, B., and Kuchta, R.D. (2000). Trends Biochem Sci. 25, 572-576.

Benna, C, et al. (2010). Curr Biol. 20, 346-352.

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 Biol. 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 Broom. 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). Scientific World Journal 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 Biol. 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 Neurol. 20, 438-446.

Steffan, J.S., et al. (2001). Nature 413, 739-743.

Takahashi, Y., et al (1996). J Biol Chem. 271, 14541-14547.

Ueberham, U., and Arendt, T. (2005). Curr Drug Targets CNS Neurol Disord. 4, 293-306.

Warrick, J.M., et al. (1998). Cell 93, 939-949.

Zoghbi, H.Y., and Botas, J. (2002). Trends Genet. 18, 463-471.

Claims

1.Use of an inhibitor of DNA replication, which DNA replication inhibitor is an inhibitor of a protein selected from primases, replication initiation factors, helicases, DNAs polymerases, DNA ligases, topoisomerases, nuclear nuclear proliferation antigens (PCNAs), replication factors A and C, 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 the replication of the DNA is an inhibitor of the replication of the DNA in the neuronal cells.

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, Cdtl, PLOA2, Timeless genes and orthologous genes. of these.

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 5, characterized in that said inhibitor of the replication of the DNA is an interfering RNA.

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, dentato-rubro-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 and Huntington's disease.