WO2017207656A1

WO2017207656A1 - Atp6ap2 inhibition for treating or preventing a tumoural and/or proliferative disorder of the central nervous system

Info

Publication number: WO2017207656A1
Application number: PCT/EP2017/063199
Authority: WO
Inventors: Matthias GROSZER
Original assignee: Universite Pierre et Marie Curie; Institut National de la Sante et de la Recherche Medicale INSERM
Current assignee: Universite Pierre et Marie Curie; Institut National de la Sante et de la Recherche Medicale INSERM
Priority date: 2016-05-31
Filing date: 2017-05-31
Publication date: 2017-12-07
Anticipated expiration: 2018-11-30

Abstract

The present invention relates to methods for screening compounds for treating or preventing a tumoural and/or proliferative disorder of the central nervous system (CNS) in an individual. Indeed, the inventors have identified a link between the expression of the ATP6AP2 gene, which encodes the (pro)renin receptor (PRR), and the occurrence of tumoural and/or proliferative disorders of the central nervous system (CNS). The inventors now provide herein experimental evidence that ATP6AP2 loss-of-function in vitro and in vivo has anti- tumour activity in tumoural and/or proliferative disorders of the central nervous system (CNS), which includes in a non-limitative manner glioblastomas. The inventions also relates to nucleic acid molecules that down regulate the expression of prorenin receptor (PRR) or a fragment thereof.

Description

ATP6AP2 INHIBITION FOR TREATING OR PREVENTING A TUMOURAL AND/OR PROLIFERATIVE DISORDER OF THE CENTRAL NERVOUS SYSTEM

FIELD OF THE INVENTION

The invention relates to screening methods and compounds, especially nucleic acids, for treating and/or preventing and/or reducing the likelihood of the occurrence of tumoural and/or proliferative disorders of the central nervous system (CNS). BACKGROUND OF THE INVENTION

Tumoural and proliferative disorders of the central nervous system (CNS) are listed in Louis et al. (2007). World Health Organization Classification of Tumours of the Central Nervous System, IARC, Lyon ; and Louis et al. (2007. The 2007 WHO Classification of Tumours of the Central Nervous System, Acta Neuropathol. ; 114 :97- 109). The World Health Organization (WHO) classification provides means to predict a response to therapy and outcome.

Indeed, tumoural and proliferative disorders of the central nervous system tend to have poor prognosis because of resistance to treatment. In particular, glioblastomas are the most frequent and aggressive adult brain tumours.

Yet, brain tumours, which are typically aggressive and heterogenenous at the cellular level, appear to have a stem cell foundation. Indeed, studies have found that human as well as experimental mouse brain tumours contain subpopulations of cells that functionally behave as tumour stem cells. Accordingly, it is believed that they share some molecular mechanisms of self-renewal with normal neural stem cells; see for reference Dirks (2010. Brain tumour stem cells; The cancer stem cell hypothesis writ large. Molecular oncology; 420- 430).

In spite of the recent advances, there remains a need in the art for relevant therapeutic compounds and thus also for screening methods of compounds for use for treating and/or preventing tumoural and/or proliferative disorders of the central nervous system (CNS) in an individual.

There also remains a need for compounds for use for treating and/or preventing tumoural and/or proliferative disorders of the central nervous system (CNS) in an individual. In particular, there remains a need for compounds which do not have side and/or non-specific effects, for instance which do not trigger significantly a stress response.

The invention herein described as for purpose to meet the aforementioned needs.

SUMMARY OF THE INVENTION

The invention relates to a method for screening compounds for treating or preventing a tumoural and/or proliferative disorder of the central nervous system (CNS) in an individual, comprising the steps of :

a) bringing a biological sample in contact with at least one candidate compound ;

b) determining the level of expression of prorenin receptor (PRR) or a fragment thereof in said biological sample; and/or determining the biological activity of PRR in said biological sample

c) selecting the said candidate compound if the said candidate compound inhibits the expression of prorenin receptor (PRR) (or the fragment thereof), or inhibits the biological activity of the PRR.

The invention also relates to a compound that down regulates the expression or biological activity of prorenin receptor (PRR) or any fragment thereof in an eukaryotic cell of an individual, for use for treating or preventing a tumoural and/or proliferative disorder of the central nervous system in said individual.

In particular, the invention relates to a nucleic acid molecule that down regulates the expression of prorenin receptor (PRR) or any fragment thereof in an eukaryotic cell of an individual, for use for treating or preventing a tumoural and/or proliferative disorder of the central nervous system in said individual.

The invention also relates to a nucleic acid molecule that down regulates the expression of prorenin receptor (PRR) or any fragment thereof in an eukaryotic cell of an individual comprising, or consisting of, at least one sequence selected from the group consisting of :

- SEQ ID N°l (CCCUUUGGAGAAUGCAGUU)

- SEQ ID N°2 (GGCAAACUCAGUGUUUGAA)

- SEQ ID N°3 (GCUCCGUAAUCGCCUGUUU)

- SEQ ID N°36 (AACUGCAUUCUCCAAAGGG)

- SEQ ID N°37 (GGAAGGCAAACUCAGUGUUUU)

- SEQ ID N°38 (AACACUGAGUUUGCCUUCCUU);

or a complementary sequence thereof. These sequences target ATP6AP2 mRNA through RNA interference.

The invention also relates to a pharmaceutical composition comprising a nucleic acid molecule or a compound as defined above for use for treating or preventing a tumoural and/or proliferative disorder of the central nervous system in an individual.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Schematic of constructs Π-ΑΤΡ6ΑΡ2 and ATP6AP2Ae4. Positions of corresponding soluble and transmembrane (TM/cyt) regions are indicated by reference to the human ATP6AP2 encoded by the mRNA NCBI sequence NM_005765. SP: signal peptide.

Figure 2: Reduction of membrane associated V-ATPase holoenzyme in HeLa cells following ATP6AP2 knockdown. Membrane-bound V1B2 subunit of the VI sector and VOal subunit of the VO sector of the V-ATPase of transfected HeLa cells were analyzed by Western blot. The band intensity was quantified and plotted as a ratio of V1B2 to VOal . mean±SEM., n=5-7 independent experiments, adjusted P-value, ANOVA (Bonferroni post- test). **P<0.01, ***P<0.001, ns: i^>>0.05. From left to right: "Scr siRNA"; "+empty vector"; "+ full-length ATP6AP2"; "+ ATP6AP2Ae4 construct"; "glucose deprivation".

Figure 3: ATP6AP2 mRNA level of expression percentage decreases in vivo in U87 glioma cell line in the presence of a siRNA targeting ATP6AP2. The ATP6AP2 is normalized in the left column at 100%. The siRNA control not targeting ATP6AP2 does not correlate with a change of the level of expression (n=5). In the presence of two siRNAs targeting ATP6AP2, the level of expression is reduced to 10% of the original value (n=5).

Figure 4: ATP6AP2 knock-down in U87 glioma cell lines correlates with decreased tumor cell growth. Three experiments are presented (n=5), over a period of 3 days (JO, Jl, J2, J3): one experiment does not bring said cells into contact with a siRNA (control). Two other experiments bring said cells into contact with a control siRNA unrelated to ATP6AP2 and, on the other hand, a siRNA targeting ATP6AP2. At day 3 (J3), the curve corresponding to the "no siRNA" experiment teaches a total number of cells (y-axis) of about 250000. The curves corresponding to the "control siRNA" and "ATP6AP2 siRNA" experiments teach respectively a total number of cells (y-axis) of about 200000 and 100000. Figure 5: Level of ATP6AP2 expression in diffuse gliomas and its prognostic value. A. Level of ATP6AP2 expression obtained from the Rembrandt database. B, Kaplan Meier plot in astrocytomas for the quartile of lower ATP6AP2 expression versus the other quartiles (p=0.0044, log-rank test, Rembrandt). C, Kaplan Meier plot in GBM for the quartile of lower ATP6AP2 expression versus the other quartiles (p=0.23, log-rank test, Rembrandt). D, Kaplan Meier plot in oligodendrogliomas for the quartile of lower ATP6AP2 expression versus the other quartiles (p=0.82, log-rank test, Rembrandt). E, Kaplan Meier plot in proneural GBM for the quartile of higher ATP6AP2 expression versus the other quartiles (p=0.005, log-rank test, TCGA).

Figure 6. Expression of ATP6AP2 in GBM. ATP6AP2 expression in U251 and U87 cell lines (RT-rtqPCR, n=4). DETAILED DESCRIPTION OF THE INVENTION

The ATP6AP2 (ATPase, H+ transporting, lysosomal accessory protein 2) gene on Xpl l .4 encodes a type I transmembrane protein mostly studied for its role as (pro)renin receptor (PRR) in the renin-angiotensin system (RAS) involved in blood pressure regulation. The ATP6AP2 protein, or PRR, is mainly composed of an extracellular (pro)renin binding domain that can be secreted after cleavage by furin, a transmembrane and a short cytoplasmic domain. The transmembrane/cytoplasmic part is present in species without renin and the extracellular domain is only conserved in vertebrates. This suggests divergent functions of the ATP6AP2 domains and that its role in the RAS was acquired during vertebrate evolution. Accumulating evidence now indicates that ATP6AP2 has important functions apart from the renin- angiotensin system.

The ATP6AP2 gene is highly expressed in the central nervous system (CNS) and human genetic studies have suggested its potential involvement in diverse neuropsychiatric disorders. Two synonymous variants in ATP6AP2 have been found to co-segregate in families with X-linked intellectual disability (XLID) and epilepsy (c.321C>T, p.D107D) and X-linked parkinsonism (XPDS) and spasticity (c.345C>T, p.S115S). Intriguingly both variants associated with these diverse disorders increase skipping of exon 4 (ΑΤΡ6ΑΡ2Δε4) that encodes part of the extracellular domain, resulting in ΑΤΡ6ΑΡ2Δε4 in -50% of ATP6AP2 transcripts. However the causative role of ATP6AP2 has been questioned, since a potential splicing variant (NM_005765.2; c.859-2A>C) resulting in a truncated protein is present in the Exome Variant Server (EVS) (13), and ΑΤΡ6ΑΡ2Δε4 transcripts are naturally occurring splice isoforms in the human brain.

WO 2013/124406 discloses the use of the secreted, extra cellular form of the soluble part of the PRR for the follow-up of a neurodegenerative disorder, or a tumoural and/or proliferative disorder associated with neurodegeneration.

Other studies suggest that the V-ATPase function of the (pro)renin receptor, or PRR, is associated with pro-proliferative effects (see Kirsch et at, 2014. The (pro)renin receptor mediates constitutive PLZF-independent pro-proliferative effects which are inhibited by bafilomycin but not genistein. International Journal of Molecular Medicine 33: 795-808).

Surprisingly, it has now been found that there is a link between the expression of the ATP6AP2 gene, which encodes the (pro)renin receptor (PRR), and the occurrence of tumoural and/or proliferative disorders of the central nervous system (CNS).

In particular, it has been found that the (pro)renin receptor is a target for inhibiting self-renewal mechanisms, such as the ones observed in tumoural and/or proliferative disorders of the central nervous system (CNS), and more particularly glioblastomas.

The inventors now provide herein experimental evidence that ATP6AP2 loss-of- function in vitro and in vivo has anti-tumour activity in tumoural and/or proliferative disorders of the central nervous system (CNS), which includes in a non- limitative manner glioblastomas.

Without wishing to be bound by the theory, the inventors are of the opinion that tumoural and/or proliferative disorders of the CNS such as glioblastoma tumour cells share some molecular mechanisms of self-renewal with normal neural stem cells. These effects could be mediated via impairment of V-ATPASE function. V-ATPASE function is involved in Wnt, Notch, EGFR and mTOR signaling pathways which are involved in oncogenic signaling and/or tumour maintenance in glioblastoma.

The inventors also propose herein that tumoural and/or proliferative disorders of the CNS such as glioblastoma tumour cells can be treated and/or prevented through the use of a nucleic acid molecule that down regulates the expression of prorenin receptor (PRR) or any fragment thereof in an eukaryotic cell, as a medicament. Advantageously, said nucleic acid molecule can be administered alone or in combination with other anti-cancer active agents, such as Temozolomide and its derivatives. The combined action of nucleic acid-mediated ATP6AP2 knock-down and anticancer agents (i.e. Temozolomide and its derivatives) is further anticipated to provide a synergistic effect, on said tumoural and/or proliferative disorders of the CNS.

In the sense of the invention, the « central nervous system » (CNS) may include, in in particular, any one of the following tissues and/or anatomic structures: cerebrum, mesencephalon, cerebellum, diencephalons, medulla oblongata, spinal cord, optic tract, superior colliculus, pituitary gland, tectospinal tract, reticular formation. If central nervous systems that are different from the above-described ones exist because of difference of species, developmental stage, abnormal development and/or disease, such tissues may also be included.

Cells in the central nervous system may non-limitatively include nerve cells, oligodendrocytes, Schwann cells, Purkinje cells, amacrine cells, retinal ganglion cells (RGC), cone cells, astrocytes, granule cells, ependymocytes, glial cells, tumour cells thereof and undifferentiated cells (stem cells) thereof.

Tumoural and proliferative disorders of the central nervous system (CNS) which are considered by the invention include any one of the tumoursd of the CNS disclosed in the World Health Organization (WHO) Classification of Tumours, and as available in Louis et al. (2007). World Health Organization Classification of Tumours of the Central Nervous System, IARC, Lyon. The 2007 WHO Classification of Tumours of the CNS is based on the consensus of an international Working Group and the contribution of international experts; it is presented as the standard for the definition.

Tumoural and proliferative disorders of the central nervous system (CNS) which are considered are also expected to be consistent with the WHO Classification of Tumours that is published in 2016.

Accordingly, tumoural and proliferative disorders of the central nervous system (CNS) may be selected from the group consisting of tumours of neuroepithelial tissue. In particular, it may be selected from the group of disorders listed in Table la,

Tumours of neuroepithelial tissue

Astrocytic

Pilocytic astrocytoma; Pilomyxoid astrocytoma; Subependymal giant cell astrocytoma; Pleomorphic xanthoastrocytoma; Diffuse astrocytoma (Fibrillary astrocytoma; Gemistocytic astrocytoma; Protoplasmic astrocytoma); Anaplastic astrocytoma; Glioblastoma; Giant cell glioblastoma; Gliosarcoma; Gliomatosis cerebri

Ependymal Subependymoma; Myxopapillary ependymoma;Ependymoma (Cellular; Papillary; Clear; cell; Tanycytic); Anaplastic ependymoma

Choroid plexus

Choroid plexus papilloma; Atypical choroid plexus papilloma; Choroid plexus carcinoma Neuronal and mixed neuronal-glial tumours

Dysplastic gangliocytoma of cerebellum (Lhermitte-Duclos); Desmoplastic infantile astrocytoma/ganglioglioma; Dysembryoplastic neuroepithelial tumour Gangliocytoma; Ganglioglioma; Anaplastic ganglioglioma; Central neurocytoma; Extraventricular neurocytoma; Cerebellar liponeurocytoma; Papillary glioneuronal tumour; Rosette-forming glioneuronal tumour of the fourth ventricle; Paraganglioma

Embryonal tumours

Medulloblastoma (Desmoplastic/nodular medulloblastoma; Medulloblastoma with extensive nodularity; Anaplastic medulloblastoma; Large cell medulloblastoma); CNS primitive neuroectodermal tumour (including Medulloepithelioma; Ependymoblastoma); Atypical teratoid / Rhabdoid tumour

Oligodendroglial

Oligodendroglioma; Anaplastic oligodendroglioma

Oligoastrocytic

Oligoastrocytoma; Anaplastic oligoastrocytoma

Other neuroepithelial tumours

Astroblastoma; Chordoid glioma of the third ventricle; Angiocentric glioma

Tumours of the pineal region

Pineocytoma; Pineal parenchymal tumour of intermediate differentiation Pineoblastoma;

Papillary tumour of the pineal region

Table la: The 2007 WHO Classification of Tumours of the Central Nervous System

Tumoural and/or proliferative disorders of the central nervous system may thus include brain (intra-cranial) tumours. Disorders of the central nervous system which are particularly considered by the invention include proliferative disorders of the CNS involving tumoural stem cells. Those tumoural and/or proliferative disorders of the central nervous system may or may not be associated with neurodegeneration. Tumoural and/or proliferative disorders of the central nervous system which are particularly considered include any tumoural and/or proliferative disorders of the central nervous system (CNS) that are CNS primitive neuroectodermal tumours.

Said tumoral and/or proliferative disorders may thus include any disorder selected from the list consisting of: glioblastomas, diffuse/infiltrating gliomas, including astrocytoma and oligodendroglioma, medulloblastoma, pineoblastoma, central primitive neuroectodermal tumour, embryonal tumour with multilayered rosettes, anaplastic ependymoma, anaplastic ganglioglioma, anaplastic pleomorphic xanthoastrocytoma. and gangliogliomas; and is preferably a glioblastoma.

According to one preferred embodiment, a « tumoural and/or proliferative disorder of the central nervous system » may be selected from the group consisting of: glioblastomas, diffuse/infiltrating gliomas (astrocytoma, oligodendroglioma), medulloblastoma, pineoblastoma, central primitive neuroectodermal tumour, embryonal tumour with multilayered rosettes, anaplastic ependymoma, anaplastic ganglioglioma, anaplastic pleomorphic xanthoastrocytoma. and gangliogliomas.

According to exemplary embodiments, the « tumoural and/or proliferative disorder of the central nervous system » is selected from a group consisting of: a glioblastoma a ganglioglioma, an astrocytoma, and an oligodendroglioma.

According to one more preferred embodiment, the « tumoural and/or proliferative disorder of the central nervous system » is a glioblastoma or a ganglioglioma.

According to its most preferred embodiment, the« tumoural and/or proliferative disorder of the central nervous system » is a glioblastoma.

Methods for screening candidate compounds

According to a first embodiment, the invention relates to a method for screening compounds for treating or preventing a tumoural and/or proliferative disorder of the central nervous system (CNS) in an individual, comprising the steps of:

b) determining the level of expression of prorenin receptor (PRR) or a fragment thereof, or the biological activity of said prorenin receptor (PRR) or fragment thereof, in said biological sample;

c) selecting the said candidate compound if the said candidate compound inhibits the expression or biological activity of prorenin receptor (PRR) or the fragment thereof. In the sense of the invention, "determining the level of expression of prorenin receptor (PRR) or a fragment thereof in a biological sample" may consist in:

(i) determining the concentration of said PRR or fragment thereof in said biological sample; and/or

(ii) determining the concentration of a nucleic acid coding for said PRR or fragment thereof in said biological sample.

In particular, step b) consists in determining the level of expression of prorenin receptor (PRR) or a fragment thereof.

In the sense of the invention, "determining the biological activity of prorenin receptor (PRR) or a fragment thereof may consist in:

(i) determining the level of activity of signaling pathways downstream of PRR through transcriptional regulation (which may include determining the concentration of a nucleic acid coding for a PRR target; and/or

(ii) determining the level of activity of signaling pathways downstream of PRR through post-transcriptional regulation (which may include determining the concentration of target polypeptides of the PRR, or determining the presence of post-translational modifications of said target polypeptides of the PRR.

Signaling pathways known to involve PRR for their activation and relevant for tumour biology are Wnt and Notch pathways, as detailled in Cruciat et al. (Science 2010 ; Hermle et al, Current Biology 2010).

In one embodiment, the biological activity of prorenin receptor (PRR) or a fragment thereof may consist in determining the membrane content of assembled V0-V1 holoenzyme complex; wherein a decrease of the assembled V0-V1 holoenzyme complex is indicative of a decrease of the biological activity of prorenin receptor (PRR).

Accordingly, a decreased membrane content of assembled V0-V1 holoenzyme complex is a specific functional readout of PRR biological activity.

The change in the membrane content of assembled V0-V1 composition can be assessed by determining the membrane ratio of the VOal subunit and of the V1B2 subunit highly expressed in the human brain (see Williamson et al. (2010. On the role of v-ATPase VOal -dependent degradation in Alzheimer disease. Commun Integr Biol 3:604-607); see also Bernasconi et al.(1990. An mRNA from human brain encodes an isoform of the B subunit of the vacuolar H(+)-ATPase. J Biol Chem 265: 17428-17431).

Protocols for cell culture, transfection, membrane fractionation and western blot are further disclosed hereafter, in the Material & Method section.

Also, a method for determining the content of assembled V0-V1 holoenzyme complex involving cell fractionation in HEK 293T cells is provided in Stransky et al. (Amino Acid Availability Modulates Vacuolar H+-ATPase Assembly. J Biol Chem. 2015 Nov 6;290(45):27360-9).

Indeed, it has been found that ATP6AP2 knockdown causes a significantly lower ratio of membrane assembled V1B2-V0al as compared to controls. Co-transfection with full- length ATP6AP2 but not with ATP6AP2Ae4 restored the V1B2-V0al ratio to control levels. Taken together these in vitro studies indicate that the lack of the exon-4 encoded ATP6AP2 domain impairs several V-ATPase mediated functions.

In the sense of the invention, an "individual" may be selected from a human or non-human individual, and preferably from a human or non-human mammal.

In the sense of the invention, a "biological sample" may be selected from any biological fluid or biopsy obtained from a human or non-human individual, and preferably from a human or non-human mammal.

A biological sample in accordance with the invention may be solid or fluid. In a non- limitative manner, a biological sample may be selected in the group consisting of: cultures, blood, plasma, serum, saliva, cerebrospinal fluid, pleural fluid, milk, lymph, sputum, semen, urine, stool, tears, saliva, needle aspirates, external sections of the skin, respiratory, intestinal, and genitourinary tracts, tumours, organs, cells, cell cultures or cell culture constituents, or solid tissue sections.

In particular, the biological sample is obtained from the CNS of a human or non-human individual, and preferably from a human or non human mammal, which may include any sample selected from the group consisting of: cerebrospinal fluid and tumours, organs, cells, cell cultures or cell culture constituents, or solid tissue sections from the CNS of said individual, as described above, such as the ones obtainable from the brain.

According to some embodiments, the biological sample is selected from: a cerebrospinal fluid, a stem cell, a stem-cell derived neuronal cell, or an extracellular medium of any neuronal cell in primary culture. According to exemplary embodiments, the biological sample is a neural stem cell or a glioblastoma cell line.

In the sense of the invention, "preventing" may also consist in "reducing the likelihood of the occurrence of a tumoural and/or proliferative disorder of the CNS.

In the sense of the invention, inhibiting" may consist in "reducing", "abolishing", and/or "preventing an increase" , by comparison to a reference value obtained from a reference sample, that is not in contact with said candidate compound.

In the sense of the invention, a "fragment of the PRR, or a nucleic acid encoding it" may comprise or consist in:

- a secreted extracellular form of the soluble part of the PRR, or a nucleic acid encoding it ; and/or

- a fragment derived from the transmembrane part of the PRR, or a nucleic acid encoding it ; and/or

- a fragment derived from the cytoplasmic part of the PRR, or a nucleic acid encoding it; and/or

- a fragment of the PRR comprising of consisting of exon 4.

The following reference peptide sequences are provided:

- a full-length human PRR of sequence of SEQ ID N°4 (NCBI Reference NM_005765.2):

- a secreted, extracellular form of the soluble part of sequence SEQ ID N°4, which corresponds to SEQ ID N°5 :

- a fragment derived from the transmembrane part of sequence SEQ ID N°4, which corresponds to SEQ ID N°6:

- a fragment derived from the cytoplasmic part of sequence SEQ ID N°4, which corresponds to SEQ ID N°7:

- a fragment of the PRR encoded by exon 4, of sequence SEQ ID N°4, which corresponds to SEQ ID N°8:

The following reference nucleic acid sequences are provided:

- a nucleic acid coding for the full-length human PRR of sequence SEQ ID N°4, which corresponds to SEQ ID N°9 (NCBI Reference NM_005765.2)

- a nucleic acid coding for the secreted, extracellular form of the soluble part of sequence SEQ ID N°4, which corresponds to SEQ ID N°10 ; - a nucleic acid coding for the fragment derived from the transmembrane part of sequence SEQ ID N°4, which corresponds to SEQ ID N°ll;

- a nucleic acid coding for the fragment derived from the cytoplasmic part of sequence SEQ ID N°4, which corresponds to SEQ ID N°12;

- a nucleic acid coding for the fragment of the PRR encoded by the exon 4, of sequence SEQ ID N°4, which corresponds to SEQ ID N°13.

In the sense of the invention, « at least 5 consecutive amino acids or nucleotides » with a reference peptide or nucleic acid sequence may include at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 25 consecutive amino acids or nucleotides (respectively) with said reference sequence.

According to a particular embodiment, the fragment of PRR may comprise or consist in a fragment having at least 5 consecutive amino acids or nucleotides with

- a fragment derived from the cytoplasmic part of the PRR, or a nucleic acid encoding it .

According to some embodiments, the fragment of PRR for which a level expression is determined in step b) comprises or consists in at least 5 consecutive amino acids or nucleotides with a fragment of the PRR encoded by the exon 4 of the PRR, or a nucleic acid encoding it. The method for screening compounds, as described herein, may comprise a step of determining the occurrence of an interaction of said candidate compound with said PRR or fragment thereof in said biological sample.

Alternatively, the method for screening compounds, as described herein, may comprise an additional step of determining the occurrence of an interaction of prorenin with said PRR or fragment thereof in said biological sample.

Accordingly, the invention further relates to a method for screening compounds for treating or preventing a tumoural and/or proliferative disorder of the central nervous system (CNS) in an individual, comprising the steps of: a) bringing a biological sample in contact with at least one candidate compound ;

bl) determining the level of expression of prorenin receptor (PRR) or a fragment thereof in said biological sample; and/or

b2) determining the occurrence of an interaction of said candidate compound with said PRR or fragment thereof in said biological sample; and/or

b3) determining the occurrence of an interaction of prorenin with said PRR or fragment thereof in said biological sample;

c) selecting the said candidate compound if, either one of the following is determined in step b):

- the said candidate compound inhibits the expression of prorenin receptor (PRR) or the fragment thereof;

- the said candidate compound interacts with said PRR or fragment thereof ;

- the said candidate compound inhibits the interaction of prorenin with said PRR or fragment thereof.

-the said candidate compound inhibits the biological activity of PRR required for the activation of signaling pathways downstream of PRR.

One example of an antagonistic peptide suitable for inhibiting, or even blocking, prorenin binding to the PRR is the pro-renin fragment PRO20 disclosed in Li et al (2014. Neuron-specific (pro)renin receptor knockout prevents the development of salt-sensitive hypertension. Hypertension 63:316-323).

Compounds for down regulating the expression of prorenin receptor (PRR)

Compounds which have been identified according to the above-mentioned methods are suitable for use for treating or preventing tumoural and/or proliferative disorder of the central nervous system in said individual.

Alternatively, those compounds can be used for the preparation of pharmaceutical compositions, or medicaments, which can be of use for treating or preventing a tumoural and/or proliferative disorder of the central nervous system in said individual.

The compounds which are particularly considered herein will be further described here-below.

Thus, according to a second embodiment, the invention relates to a compound that down regulates the expression of the prorenin receptor (PRR) or a fragment thereof in a eukaryotic cell of an individual, for use for treating or preventing a tumoural and/or proliferative disorder of the central nervous system in said individual.

In the sense of the invention, a "compound that down regulates the expression of the prorenin receptor (PRR)" is a compound which, when brought into contact with an eukaryotic cell from said individual is able to decrease the expression of the prorenin receptor (PRR) over a reference eukaryotic cell that is not brought into contact with said compound under the same conditions.

Down-regulation of the expression of the prorenin receptor (PRR) or any fragment thereof may be achieved by:

(i) decreasing the concentration of said PRR or fragment thereof; and/or

(ii) decreasing the concentration of a nucleic acid coding for said PRR or fragment thereof, which may include decreasing the expression of the nucleic acid and/or decreasing the stability of the nucleic acid.

According to some embodiments, such compound is in the form of a polypeptide or of a nucleic acid.

According to a particular embodiment, the invention relates to a nucleic acid molecule that down regulates the expression of the prorenin receptor (PRR) or a fragment thereof in an eukaryotic cell of an individual, for use for treating or preventing a tumoural and/or proliferative disorder of the central nervous system in said individual.

When the compound that down regulates the expression of the prorenin receptor

(PRR), or fragment thereof, is a nucleic acid, it may be either a single-stranded or a double- stranded nucleic acid.

Still, when the compound that down regulates the expression of the prorenin receptor (PRR), or fragment thereof, is a nucleic acid, it may also be a deoxy-ribonucleic acid (DNA) or a ribonucleic acid (RNA).

Thus, when the compound that down regulates the expression of the prorenin receptor (PRR), or fragment thereof, is a nucleic acid, it may be selected from a group consisting of single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA).

Examples of nucleic acids which down-regulate the expression of the PRR, or a fragment thereof, in an eukaryotic cell may be selected from the group consisting of: ribozymes, antisense nucleic acids, short interfering nucleic acids (siNA), short-interfering RNAs (siRNAs), micro-RNAs (miRNA), short hairpin RNAs (shRNA), short interfering oligonucleotide, short interfering substituted oligonucleotide, short interfering modified oligonucleotide.More preferably, the compound that down regulates the expression of the prorenin receptor (PRR), or fragment thereof, is an interfering RNA, such as a siRNA or a miRNA; and most preferably a siRNA.

The term "siRNA ", or "small interfering RNA" is known in the Art and generally refers to small double-stranded RNAs of about 20 to 25 nucleotides, which encompasses 20, 21, 22, 23, 24 or 25 nucleotides.

The term "miRNA ", or "micro-interfering RNA" is known in the Art and generally refers to small single-stranded RNAs of about 19 to 25 nucleotides, which encompasses 19, 20, 21, 22, 23, 24 or 25 nucleotides

According to an exemplary embodiment, the invention relates to a nucleic acid molecule for its use as defined above, wherein said nucleic acid molecule is a an interfering RNA, in particular a siRNA, comprising (or even consisting of) at least one sequence selected from the group consisting of : SEQ ID N°l to 3, or SEQ ID N°36 to 38; or a complementary sequence thereof.

According to a third embodiment, the invention relates to a nucleic acid molecule that down regulates the expression of prorenin receptor (PRR) or any fragment thereof in an eukaryotic cell of an individual; comprising (or even consisting of) at least one sequence selected from the group consisting of : SEQ ID N°l to 3, or SEQ ID N°36 to 38; or a complementary sequence thereof.

This nucleic acid molecule is preferably an interfering RNA, in particular a siRNA.

Compounds, such as nucleic acids, as defined above, are considered both in an isolated form, or in a pharmaceutical composition.

According to a fourth embodiment, the invention relates to a pharmaceutical composition comprising a compound as described above, in particular a nucleic acid as described above, for use for treating or preventing a tumoural and/or proliferative disorder of the central nervous system in an individual.

According to some embodiments, a compound, nucleic acid or pharmaceutical composition as described above is suitable for enteral, parenteral, intra-muscular, cutaneous, sub-cutaneous, intra-spinal, intra-cerebral and/or intracerebroventricular administration.

According to some embodiments, a compound, nucleic acid or pharmaceutical composition as described above is injectable and/or in a sterile form. According to some embodiments, a compound, nucleic acid or pharmaceutical composition as described above is administered in combination with an additional drug, such as temozolomide and salts thereof.

In particular, and in the case of glioblastoma, it is administered in combination with temozolomide and salts thereof.

EXAMPLES

1. MATERIAL & METHODS

Ethics approval.

Informed consent was obtained from the parents of the patient carrying the

ATP6AP2 (c.321C>T) variant and the study was approved by the Institutional Review Committee (IRB#26) of Self Regional Healthcare, Greenwood, South Carolina, USA. Informed consent was obtained from the parents of the patient carrying the ATP6AP2 [c.301- 11 301-lOdelTT] variant and the study was approved by the Research Ethics Board of the Mount Sinai Hospital, Toronto, Ontario, Canada. The parents gave informed consent for blood sampling and skin biopsy to generate iPSC lines.

Sequencing of patient DNA.

The intronic ATP6AP2 variant in the patient was identified by using the XLID Next-Gen panel sequencing screen (Ambry Genetics, Aliso Viejo, CA 92656) and verified by Sanger sequencing (Charles E. Schwartz, Greenwood Genetic Center, Greenwood, SC 29646, USA). The Ambry XLID Next-Gen Panel™ targets detection of mutations in 81 genes by sequencing of all coding domains plus at least 10 bases into the 5' and 3' ends of all introns. Generation and characterization of iPSCs.

Fibroblasts from ATP6AP2 [c.301-11 301-lOdelTT] patient were reprogrammed into inducible pluripotent stem cells line PB41 (iPSCs), using non- integrating CytoTune™ - Sendai viral vector kit (Life Technologies, A13780) according to manufacturer's instructions with slight modifications. Briefly, reprogramming was achieved by overnight transduction of 1 x 10⁵ fibroblasts in low-serum containing fibroblast medium (FibroGRO™-LS, Millipore) at a multiplicity of infection of 6 (MOI6). Forty-eight hours later, transduced cells were passaged onto freshly ES-qualified Matrigel™ (BD-Biosciences) coated 60 mm dish in FibroGRO-LS medium. The next day, medium was switched to Nutristem medium (Miltenyi Biotec) and changed every day until the emergence of reprogrammed colonies. IPS colonies were identified under a stereomicroscope (Lynx, Vison Engineering) at day 25 post-transduction and were manually picked and plated onto Mitomycin-C (Sigma) inactivated mouse embryonic fibroblasts in KOSR medium composed of DMEM/F12 culture medium, supplemented with 20% KnockOut Serum Replacement, 0.1 mmol/L non-essential amino acids, 1 mmol/L L- glutamine, 0.1 mmol/L 2-mercaptoethanol, penicillin/streptomycin (all of them from Life Technologies) and 12.5 ng/mL recombinant human basic fibroblast growth factor (Miltenyi Biotec). Control iPSC was obtained from reprogramming of human foreskin fibroblasts (FibroGRO™ Xeno-Free Human Foreskin Fibroblasts, Millipore) with Stemgent mRNA Reprogramming kit (Miltenyi Biotec) according to manufacturer's instructions. The iPS colonies were further expanded in KOSR medium onto inactivated MEF. All the cultures were performed at 37°C in a 5% C02 atmosphere. The iPSC lines were characterized by FACS analysis for the expression of pluripotent stem cell surface markers. Briefly, 105 cells were stained with a combination of BD Horizon™ V450-conjugated mouse anti-human SSEA4 (Clone MC813-70; BD Biosciences), fluorescein isothiocyanate (FITC)-conjugated mouse anti-human HESCA1 (clone 051007-4A5; Millipore), phosphatidylethanolamine (PE) - conjugated rat anti-mouse SSEA3 (clone MC631 ; BD Biosciences) and Alexa Fluor® 647- conjugated mouse anti-human TRA-1-60 (clone TRA-1-60; BD Biosciences) according to the manufacturer's recommendations. Cells were analyzed on a MACSQuant flow cytometer (Miltenyi Biotech) using the MACSQuantify software. The genomic integrity was assessed by karyotyping according to standard procedures. Following reprogramming, fl-ATP6AP2 and ΑΤΡ6ΑΡ2Δε4 transcripts were present. Pluripotency of the iPSC line was assessed by teratoma formation assays. Six week-old NSG mouse was subjected to intramuscular injection of 2xl0⁶ to 3xl0⁶ iPSCs. After 8 weeks, teratomas were dissected, fixed in 4% paraformaldehyde and samples embedded in paraffin and stained with hematoxylin-eosin.

Neuronal differentiation of iPSCs.

Differentiation of iPSCs into cortical neurons was performed as described in Pasca et al. (201 1. Using iPSC-derived neurons to uncover cellular phenotypes associated with Timothy syndrome. Nat Med 17: 1657-1662). Briefly, iPSC colonies were detached by treatment with type IV collagenase and kept in suspension in low attachment plates (Corning) as embryoid bodies (EBs) during five days in iPSC culture media without FGF2 containing 10 μΜ SB421542 (Sigma) and 20 μΜ Dorsomorphin (Sigma). EBs were then plated in polyornithine/laminin-coated dishes and kept in culture for seven extra days in neural precursor media (NPC: Neurobasal, B27 minus Vitamin-A (Gibco), FGF2 (20 ng/μΕ) and EGF (20 ng/μΐ.) until neural rosettes were observed. These rosettes were then picked manually and dissociated into single cell suspension using trypsin (TrypLETM, Gibco) and plated at a density of 1.5 x 10⁵ cells/mm2 in neuronal differentiation media (NDM media: Neurobasal, B27 minus Vitamin-A, BDNF (10 ng/jiL), GDNF (10 ng jiL) and cAMP (100 μΜ). Western blots for p62 and LC3B were performed according to guidelines for autophagy assays in Barth et al. (2010. Autophagy: assays and artifacts. J Pathol 221: 117-124).

Calcium Imaging.

The iPSC-derived neurons at 45DIV were bulk loaded with 5 μΜ of Fluo4-AM (Life technologies) in culture medium for 15 min at 37°C. Then the culture medium was removed and replaced by an imaging medium: MEM medium, 4 mM sodium bicarbonate, 20 mM HEPES, 2 mM GlutaMAX, 33 mM D-glucose, B27 and N-2 serum. During imaging, iPSC were perfused at a rate of 1 mL/min with imaging medium at 32°C. Images were obtained with an upright microscope DM6000CFS (Leica Microsystems, Wetzlar, Germany) equipped with a 25 x 0.95 numerical aperture water-immersion objective and a CCD camera (DFC 360 FX, Leica Microsystems). Illumination was a short arc lamp HXP (Osram); exposure times were 0.1-0.2 s. Excitation and dichroic filters were D480/40 nm and 505 nm. Signals were acquired behind an emission filter (527/30 nm). Recorded fluorescence movies (1 image/s during 5 min) were first corrected for movement artifacts using the StackReg plug-in for ImageJ. The calcium signal for each detected cell was extracted from stable portions of movies. In these traces, the large deflections indicate calcium events that correspond to the increase of neuronal activity. The onset of each deflection was marked and used for building RASTER plot (Matlab). Fibroblast cell culture.

Explants of 3-mm dermal biopsies were minced and placed in a 60-mm tissue culture dish under a sterile coverslip held down by sterilized silicon grease. Fibroblast medium [Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal calf serum (FCS), Glutamax™, and penicillin/streptomycin (Invitrogen, Carlsbad, CA) was added, and dishes incubated at 37°C in a humidified 5% C02 atmosphere with media exchange every 5 days. Fibroblast outgrowths were harvested by trypsinization, expanded in a T25 flask in fibroblast medium, and allowed to reach -90% confluence prior to freezing or splitting for reprogramming as described below. For reprogramming, fibroblasts were used within the first three passages from biopsy or within one passage after a thawing. Electron Microscopy

Cells were fixed with 4% PFA (paraformaldehyde) and 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB) (1 h) and post-fixed in Os04 (40 min) at 4°C. Samples were dehydrated in an ascending series of ethanol, and embedded in epoxy resin. Semi-thin sections (0.5 μηι) were stained with toluidine blue and viewed with a Provis Olympus Microscope. Images were acquired with a Coolsnap CCD camera. Ultra-thin sections (40 nm thick) were mounted in 200 meshes, cut and double stained with uranyl acetate and lead citrate prior to observation with a Philipps (CM- 100) electron microscope. Digital images were obtained with a CCD camera (Gatan Orius). Morphometric analysis was performed with the software Digital Micrograph on two different experiments of each genotype. For lysosomal diameter, lysosomes were identified by their morphology as described in Wei et al. (2007. Enhanced lysosomal pathology caused by beta-synuclein mutants linked to dementia with Lewy bodies. J Biol Chem 282:28904-28914) and their diameter measured in 10 neurons per genotype.

Animals

Mouse strains were on C57BL/6 genetic background and have been described earlier: Atp6ap2 flox (10), Emxl-Cre (60) and Cam KII Cre (61, 62). All procedures were designed to minimize animal suffering and carried out in accordance with the European Guidelines for the care and use of experimental animals of the European Economic Community (2010/63/UE) and the French National Committee (87/848) for the use of laboratory animals. The study protocol was approved by the Institutional Animal Care Committee, license number 01513.01. Behavior tests

Open field test and contextual fear conditioning were performed as described in Kiyasova et al. (2011. A genetically defined morphologically and functionally unique subset of 5-HT neurons in the mouse raphe nuclei. J Neurosci 31 :2756-2768). The open field test was conducted in a brightly illuminated square arena. Mice were placed in the center of the arena and allowed to explore the area for 10 min. Behavior was tracked using a ceiling-mounted camera (Panasonic WV BP332). For contextual fear-conditioning, mice were placed into a conditioning chamber and allowed to habituate for 3 min, followed by three consecutive foot shocks (1.0 mA, 50 Hz, 1 s) separated by 1 min. Animals remained in the chamber for 1 min after the delivery of the last foot shock. Testing occurred 24 h after in the same chamber, and freezing was scored for a total of 5 min. Sessions were recorded and freezing was manually scored blinded for genotype. Freezing was defined as complete absence of movement, except for respiration. Data were expressed as percentage of time freezing. Immunohistochemistry

Sections of 40 μηι (P15, 6 months) and 60 μηι (E12, E13, E14) were prepared using a VT1000S vibratome (Leica Biosystems) and maintained at -20°C in cryoprotectant tissue collecting buffer (30% ethylene glycol, 30% glycerol in 0.024 M phosphate buffer, PB). For frozen section, brains were cryostat-sectioned into 5-10 μηι slices (Leica CM 1800, Leica Biosystems). Nonspecific binding was blocked by pretreatment with 5% normal goat serum-in PBS containing 0.2% gelatin. The sections were incubated in primary antibodies overnight at 4°C in PBS 0.2% gelatin or 0.4% saponin. The list of antibodies is provided in Table 2. BrdU and Ki67 staining was performed as described in Chenn et al. (2002. Regulation of cerebral cortical size by control of cell cycle exit in neural precursors. Science 297:365-369). Alexa 488 or Alexa 555 fluorescent secondary antibodies (Invitrogen) were used for Leica laser-scanning two-photon microscopy (Leica TCS SP5, Leica Biosystems, Wetzlar, Germany) and secondary antibodies (Jackson Immuno Research) were conjugated to Atto 565 (ATTO-TEC, Siegen, Germany) and DY 485 (Dyomics, Jena, Germany) and used for two-color STED microscopy on a custom built setup as in Lauterbach et al. (2013. STED microscope with spiral phase contrast. Sci Rep 3 :2050).

Antigen Company Catalog number Host IF FACS WB

ACTB Santa Cruz sc-47778 mouse 1 :6000

ATP6AP2 Dr G Nguyen rabbit 1 : 100

ATP6AP2 Sigma-Aldrich HPA003156 rabbit 1 : 1000

ATP6V1B2 Dr CA Wagner rabbit 1 : 1000

ATP6V0al Synaptic Systems 109 002 rabbit 1 : 1000

BrdU AbD serotec OBT0030 rat 1 :400

CASP3 BD Pharmingen 559565 rabbit 1 :500

CDC42 Santa Cruz sc-8401 mouse 1 : 100

CDH2 (N-cadherin) ZYMED 33-3900 mouse 1 :250

CTNNB1 Sigma-Aldrich C2206 rabbit 1 :500 EGFR Santa Cruz sc-03 rabbit 1 : 1000

HESCA-1 (MC813-70) Millipore FCMAB111F mouse 1 :10

LAMP1 BD Pharmingen 553792 rat 1 : 100

LC3B Cell signaling 2775 rabbit 1 :400 1 : 1000

Mki67 (Ki-67) Novocastra NCL-Ki67p rabbit 1 : 1000

MPP5 (PALS1) Millipore 07-708 rabbit 1 :200

mTOR Cell signaling 2983 rabbit 1 :200

PARD3 (PAR3) Millipore 07-330 rabbit 1 :300

PAX6 Covance PRB-278P rabbit 1 :500

pH3 Millioore 06-570 rabbit 1 :200

p-mTOR (Ser2448) Cell signaling 5536 rabbit 1 :50

PROM1 (CD133) Millipore MAB4310 rat 1 :200

RAB5A Sigma-Aldrich R7904 mouse 1 :500

RAB7 Sigma-Aldrich R8779 mouse 1 :500

SQSTM1 (p62) Millipore MABC32 mouse 1 :750

SQSTM1 (p62) Abeam ab91526 rabbit 1 : 1000

SSEA-3 BD Pharmingen 560237 mouse 1 :10

SSEA-4 BD Horizon 561156 mouse 1 :10

TFEB Bethyl A303-673A rabbit 1 :750

TRA-1-60 BD Pharmingen 560122 mouse 1 :10

TUBA1A Sigma-Aldrich T5168 mouse 1 :5000

TUBB3 (TUJ1) Covance MMS-435P mouse 1 :2000

Table 2: Sources and working dilutions of antiboc lies

Cell culture and transfection

HeLa cells were cultured in DMEM (Invitrogen) containing 10% FCS. Constructs, scramble and siRNAs targeting the 3' -UTR (Dharmacon, Rockford, IL) were transfected into HeLa cells using lipofectamine 2000 (Life Technologies) according to the manufacturer's instructions.

Vesicular pH measurement, fluorescent microscopy and image processing. Vesicular H was determined as described (65). Briefly, HeLa cells were seeded in 8-well Labtek II chambers (Thermo Fisher Scientific) and transfected with siRNA with or without rescue constructs. Twenty-four hours later, cells were incubated in 1 mg/mL Lysosensor Yellow/Blue dextran (Invitrogen) for 1 h, with or without 100 nM bafilomycin. Cells were then washed twice in PBS. For each experiment, a pH standard curve was obtained by treating the cells with clamp buffers at pH 3.5 to 7.5. Cells were equilibrated in clamp buffer pH 3.5-7.5 (5 mM NaCl, 115 mM KC1, 1.2 mM MgS04, 25 mM MES, pH 3.5-7.5) followed by washing in clamp buffer supplemented with 25 μΜ monensin and 10 μΜ nigericin (Sigma) for 20 min before imaging. All equilibration steps were performed in a 37°C, 5% C02 incubator. Imaging was performed on a heated stage (37°C). Cells were excited with attenuated UV light (357-373 nm) and observed in blue (Wl; 417-483 nm) and yellow (W2; 490-530 nm) region of the spectra. Images were collected on a Leica DMIRBE, using a 63x/1.25 NA objective over a period of 5 min. Sixteen-bit images were processed to subtract background fluorescence, and then ratio (W1/W2) images were generated using the image calculator function in ImageJ. Regions of interest (ROIs) corresponding to vesicles were generated using W2 and applied to ratio images to determine the W1/W2 ratio value of the vesicles for each pH clamp buffer. A standard curve was generated in Prism to assign a predicted pH for W1/W2 values. Epidermal growth factor receptor (EGFR) degradation.

EGFR degradation was studied as described in Endo et al. (2008. Regulation of clathrin- mediated endocytosis by p53. Genes Cells 13:375-386 using a HeLa cell line with stable knock-down of ATP6AP2 (ATP6AP2 HeLa SilenciX, Tebu-bio) cultured in DMEM 10% FCS and supplemented with Hygromycin according to manufacturer instructions. Briefly, control and ATP6AP2-KD HeLa (SilenciX, Tebu-bio) were cultured in 6 well plates at 90% confluence with 10%) FCS/DMEM containing 125 mg/mL hygromycin. Cells are starved with 0.1% FCS/DMEM (without antibiotics) for 20 h and then pre-incubated with 10 mg/mL cycloheximide for 30 min and stimulated with 100 ng/mL recombinant human EGF (236-EG- 200; R&D system) for 0, 30, 60, 120, 180, 240 min, in triplicate. Cells are then washed with ice cold PBS and lysed with lysis buffer (20 mM Hepes-KOH pH7.4, 100 mM KC1, 0.5 mM EDTA, 10 mM NaF, 1% Triton X-100, 1 mM Na₃V0₄, 10 mM Na4P207, 0.1 mM Na₂Mo0₄, β-glycerolphosphate, protease inhibitor cocktail (Complete; Roche-diagnostics) and scraped. Cell lysates were analyzed by 7.5% SDS-PAGE and Western blotting with anti-EGFR (sc-03, Santa Cruz). Cloning and expression

Expression vectors for either human fl-ATP6AP2 cDNA or ΑΤΡ6ΑΡ2Δε4 were generated from the patient with ATP6AP2 variant c.321C>T (p.D107D) (OMIM #300423) (67) and ligated into the KpnI/EcoRI site in pcDNA3.1 (+) vector (Invitrogen, Carlsbad, CA) (Table 3). The schematic of A-ATP6AP2 and ATP6AP2Ae4 is provided in figure 1.

Table 3: PCR primers

Vesicle fusion and live cell imaging

Endosome-lysosome fusion assays were adapted from Bright et al. (2005. Endocytic delivery to lysosomes mediated by concurrent fusion and kissing events in living cells. Curr Biol 15:360-365). Briefly, HeLa cells were seeded in 8-well Labtek II chambers and trans fected with siRNA with or without rescue construct. Then cells were loaded with 50 nM Lysotracker red (Life Technologies) for 4 h followed by incubation in conjugate-free medium for 20 h. Cells are then loaded with dextran Oregon green 488 (10,000 MW, anionic, fixable; Life Technologies) for 10 min followed by a 5 min chase in conjugate- free C02- independent medium (Invitrogen). Cells were washed 3 times with PBS, transferred to the heated stage and spinning-disk images were acquired using a 63x/0.15 NA objective on a Nikon Eclipse Ti microscope, equipped with an Evolve 512/EM-CCD-camera (Photometries) and a CSUX1-A1 (Yokogawa) confocal scanner. Fluorescence was excited with a 491 nm laser and detected with a 525/39 nm filter for Oregon green 488. A 561 nm laser and 605/64 nm filter were used for Lysotracker red. MetaMorph software (Molecular Devices) was used to collect the data and ImageJ to assemble the movies.

Membrane fractionation and Western blotting

To prepare membrane fractions, HeLa cells were homogenized with a Teflon potter in sucrose buffer and the postnuclear supernatant was fractionated into cytosolic and membrane fractions by ultracentrifugation (60 min, 100,000xg) as described in Trombetta et al. (2003. Activation of lysosomal function during dendritic cell maturation. Science 299: 1400- 1403). Alternatively cell lysate was obtained by treating cells with lysis buffer (Cell signaling) containing protease and phosphatase inhibitors. Total protein was separated on 10% or 16% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were blocked with 5% non-fat dry milk or 5% BSA in 20 mM Tris-HCl pH 7.4, 150 mM NaCl with Triton X-100 for 30 min at room temperature and incubated overnight with primary antibodies at 4°C. Membranes were incubated with alkaline-phosphatase conjugated secondary antibodies (1 :5,000; Jackson ImmunoResearch) for 1 h at room temperature. AttoPhos® AP Fluorescent Substrate System (Promega) was used for detection and quantification of bands was performed using ImageJ software. The primary antibodies used are listed in Table 2. Statistics

Pv (version 2.14.0) (http://www.R-project.org) and graph pad prism software has been used for statistical analyses. Data normality was verified by Shapiro- Wilk test before performing parametric or nonparametric tests, as described in Royston (1982. Algorithm AS 181 : the W-test for normality. Applied statistics 31 : 176- 180).

2. RESULTS

EXAMPLE 1 : ATP6AP2Ae4 impairs V-ATPase activity and function 1) ATP6AP2 is required for cortical development.

A de novo intronic ATP6AP2 variant was identified in a boy with X-linked intellectual disability (XLID) and fulminant early postnatal neurodegeneration (also referred herein as 'patient 7"). Candidate gene sequencing has revealed a deletion of two conserved nucleotides in intron 3 in ATP6AP2 [c.301-11 301-lOdelTT]. The ATP6AP2 variant is predicted to disrupt a branch point motif at position c.301-14 expected to increase exon 4 skipping. Exon-4 encodes for a part of the N-terminal extracellular/intravesicular domain, outside of the initially identified V-ATPase interaction domain. Indeed, RT-PCR of patient fibroblasts showed 20% full-length-(fl)ATP6AP2 and 80% ATP6AP2Ae4 transcripts.

These results suggested neurodevelopmental deficits or/and neurodegenerative processes that prominently affect cortical regions in these patients. We therefore studied neural Atp6ap2 functions in cortical development. We first analyzed its cortical expression in embryonic day 12 (El 2) mice. The subcellular distribution of Atp6ap2 suggested its involvement in radial glial cells (RGC) polarity and ultimately cell fate choice, consistent with recent data in retinal progenitors

We also generated mutant mice carrying a conditional deletion (Atp6ap2^Iox/y;Emxl-Cre ^~) in order to assess ATP6AP2 functions in corticogenesis. Rescue experiments in Pals 1 mutants suggested a role for mammalian target of rapamycin (mTOR) kinase signaling in coupling apical complex dysfunction in progenitors and neuronal cell death.

We studied the expression of proteins involved in RGC polarity in Atp6ap2 mutants. Immuno-labeling revealed that Palsl, Par3, Proml (prominin, CD133) or Cdc42 as well as adherens junction proteins Cdh2 (N-Cadherin) and Ctnnbl (β-catenin) were largely absent in Atp6ap2 cKO. The failure to detect a broad range of apical proteins in Atp6ap2 cKOs suggested severe deficits in fundamental cellular processes such as protein trafficking, recycling or degradation interfering with multiple signaling pathways. Lysosomes are terminal compartments of endocytosis and autophagy and are signaling hubs to coordinate cellular responses to changes in nutrients availability via mTOR signaling.

We studied the involvement of ATP6AP2 in lysosomal functions in the developing cortex. These results indicate induction of autophagy and accumulation of autophagosomes but deficient protein degradation in autophagolysosomes in mutants. Taken together these results provide evidence that ATP6AP2 is a key regulator of V-ATPase functions during corticogenesis. ATP6AP2 deficiency may lead to dysfunctions of several V-ATPase dependent cellular processes such as vesicular acidification, protein degradation and mTOR signaling.

To test whether behavioral abnormalities were correlated with morphological signs of lysosomal pathway dysfunction, we performed immunostaining on cortex and hippocampus in six month-old mutants. Similar to observations in the embryonic brain, Lampl staining revealed an increased accumulation of enlarged lysosomal compartments clustered in the perinuclear region. We further examined autophagosomes by staining for LC3B and its interacting partner p62/SQSTMl that targets ubiquitinated proteins to autophagosomes. We found increased perinuclear staining and a significantly higher number of LC3+ and p62/SQSTMl+ punctae. However, we did not detect fulminant neuronal degeneration or increased immunoreactivity of the apoptosis marker activated-caspase 3, suggesting that mature as compared to developing neurons are less sensitive to ATP6AP2 deficiency.

2) Abnormal 'in vitro corticogenesis', impaired proteostasis, activity and survival of patient iPSC-derived neurons.

To study whether increased exon 4 skipping causes lysosomal dysfunction during human cortical development we generated iPSCs from patient 1.

When sequentially cultured in media containing different growth factors, iPSC recapitulate key steps of in vivo corticogenesis. Control iPSCs organized after 20 days in vitro (DIV) into characteristic sphere-like structures termed neural rosettes resembling the cortical neuroepithelium, with PAX6⁺ progenitors at the luminal surface and TUJ1⁺ neurons in the outer layer. Patient-derived neural rosettes showed TUJ1⁺ cells interspersed in PAX6⁺ progenitor zone, suggesting premature and/or ectopic differentiation. Phospho-histone 3 immunolabelling indicated no differences in proliferation potential of progenitor cells between patient and control. Accordingly, PAR3, CDH2 and CTNNB1 expression did not differ from controls. We used the acidotropic dye LysoSensor yellow/blue Dextran to quantify vesicular acidification across a broad (3.0-9.0) pH range (see Wolfe, D.M., Lee, J.H., Kumar, A., Lee, S., Orenstein, S.J., and Nixon, R.A. 2013. Autophagy failure in Alzheimer's disease and the role of defective lysosomal acidification. Eur J Neurosci 37: 1949-1961). LysoSensor measurements showed a significantly increased mean vesicular pH in patient iPSC-derived cortical neurons suggesting an impaired V-ATPase function; thus highlighting abnormal in vitro corticogenesis.

We further studied early developing neurons. Electron microscopy of these neurons showed excessive accumulation of large vacuoles containing diverse material resembling late autophagic compartments and increased lysosomal diameter. Western blots of the patient iPSC derived neurons showed significant increases of p62 and LC3B as well as of the autophagy-activated form LC3B-II, suggesting a feedback up-regulation of autophagosome formation. With progressing time of neuronal differentiation, cell death of patient iPSC- derived neurons significantly increased when compared to control, as confirmed by activated caspase 3 immunostaining. The survival of developing cortical neurons is critically dependent on early spontaneous activity. We measured spontaneous intrinsic neuronal activity in developing iPSC derived cortical neurons at 45 DIV using calcium imaging.

We found that the number of spontaneously active neurons as well the frequency of their activity was significantly lower in patient than in control iPSC-derived cultures. Thus ATP6AP2 deficiency may impair early spontaneous activity which in turn may contribute to the fulminant neurodegeneration observed in the patient.

3) ATP6AP2Ae4 impairs V-ATPase activity and functions

Our studies on patient iPSC derived neurons thus provide evidence for the requirement of the exon-4 encoded domain in V-ATPase functions.

In order to gain further insights into ATPase dysfunctions due to the lack of the exon-4 encoded ATP6AP2 domain, we studied HeLa cells using siRNA mediated knockdown (commercial siRNAs from ThermoScientific) yielding -75% reduction of protein levels. LysoSensor assays showed that ATP6AP2 knockdown increased median vesicular pH from 6.01 to 6.42 with a notable decrease of the more acidic vesicles (pH 3.5-4.5) likely representing lysosomes. Co-trans fection with fl-ATP6AP2 but not with ΑΤΡ6ΑΡ2Δε4 restored pH to control levels, confirming that the exon 4-encoded domain is necessary for vesicular acidification.

We next assessed whether this acidification deficit is associated with impaired degradation of endocytosed proteins. We analyzed ligand- induced degradation of epidermal growth factor receptor (EGFR) using a HeLa cell line with stable ATP6AP2 knock-down and found that ATP6AP2 knockdown causes delayed the decay of EGFR. Although decreased activity of lysosomal hydrolases due to impaired vesicular acidification may account for this delay, inhibition of V-ATPase activity may also impair endosomal- lysosomal fusion. We used time-lapse confocal microscopy to study fusion events following ATP6AP2 knockdown. LysoTracker Red was used to label lysosomal compartments whereas endosomes were identified via uptake of a pulse of Oregon green 488 dextran, the subsequent appearance of yellow dots indicated fusion events. Whereas control cultures displayed a characteristic combination of kissing and fusion events, ATP6AP2 knockdown surprisingly caused a rapid and significant increase in fusion events especially in large perinuclear clusters. Co- transfection with fl-ATP6AP2 but not with ΑΤΡ6ΑΡ2Δε4 restored fusion events to control levels. In particular, reduction of membrane associated V-ATPase holoenzyme was observed in Hela cells following ATP6AP2 knockdown (see figure 2).

Taken together these in vitro studies indicate that the lack of the exon-4 encoded ATP6AP2 domain impairs several V-ATPase mediated functions.

4) Conclusion

We found that ATP6AP2 is a key accessory protein for V-ATPase functions in the

CNS and essential for stem-cell self-renewal and neuronal survival. These findings have implications beyond XLID for more common disorders.

The vacuolar H+-adenosine triphosphatases (V-ATPases) are proton pumps present on endomembranes of all cells and acidify intracellular compartments, which is critical for numerous cellular processes, such as protein trafficking, maturation, recycling or degradation. In addition V-ATPases have been also involved in acidification-independent roles such as membrane fusion or secretion.

In lysosomes, V-ATPases regulate the optimal acidic pH for diverse enzymes to degrade macromolecules delivered from endocytic and autophagic pathways. In addition lysosomal V-ATPases function as docking platform and amino-acid sensors to regulate the activity of the mechanistic target of rapamycin complex 1 (mTORCl), a master regulator of cell growth and autophagy. Although dysruptions of lysosomal V-ATPase-mediated functions may impair protein homeostasis (proteostasis) in different tissues, they are frequently linked to neurodegenerative disorders.

Collectively the patient data suggest that reduced V-ATPase activity due to ATP6AP2 deficiency may have an interesting safety margin, affecting the brain while being more tolerated in other tissues. The animal studies suggest, that while Atp6ap2 knockout rapidly inhibits stem-cell self-renewal, the more mature brain appears less sensitive. This is relevant for the development of V-ATPase inhibitors for brain cancers, which rely heavily on pH regulation for growth and invasion.

Therefore the identification of V-ATPase associated molecules regulating its activity in the CNS will deliver attractive targets for drug development.

EXAMPLE 2: Assessment of anti-tumour activity of ATP6AP2 loss-of- function in glioblastoma.

The anti-tumour activity of ATP6AP2 loss-of-function can be assessed in brain tumours according to any one of the following protocols.

1) Method 1 : Loss of function of ATP6AP2 in vivo in murine glioma by tamoxifen-induced cre-mediated knock out.

Step 1 : Conditional KO mouse lines are generated by interbreeding floxed ATP6AP2 mice and mice carrying a tamoxifen-inducible ubiquitary Cre recombinase, as disclosed in Hayashi & McMahon (2002. Efficient recombination in diverse tissues by a tamoxifen- inducible form of cre: a tool for temporally regulated gene activation/inactivation in the mouse. Dev Biol 244(2):305-18).

Step 2 : oncogenic transformation is induced by cre-independent lentivirus : shP53 H-Ras AKT according to Friedmann-Morvinski et al. (2012. Dedifferentiation of neurons and astrocytes by oncogenes can induce gliomas in mice. Science 338, 1080-1084) and Marumoto et al. (2009. Development of a novel mouse glioma model using lentiviral vectors. Nat Med 15, 110-116).

- Option A : in vitro ; by generating embryos and culture of embryonic neural stem cells and then transfecting them with the lentivirus;

- Option B : in vivo ; by injecting lentivirus in mouse brain ; follow-up of the injected mouse and then serial transplantation of induced brain tumour in nude mice.

Step 3 : graft in nude mice followed by tamoxifen induction in control and cre- dependent ATP6AP2 tumours. 2) Method 2 : Loss of function of ATP6AP2 in vitro in human GBM cell lines.

Human glioblastoma (GBM) cell lines from Gliotex biobank with known genotype are provided (i.e. with EGFR amplification). Cell death is measured using CellTiter-Glo luminescence assays (Promega) after ATP6AP2 siRNA transfection, optionally in the presence of an additional chemotherapy agent, such as temozolomide. See Verreault et al. (Preclinical Efficacy of the MDM2 Inhibitor RG7112 in MDM2-Amplified and TP53 Wild-type Glioblastomas. Clin Cancer Res. 2016 Mar 1;22(5): 1185-96).

EXAMPLE 3: in vivo evidence that a siRNA targeting ATP6AP2 lead to decreased tumor cell growth and proliferation in a U87 glioma cell line.

We tested the efficiency of the siRNA targeting of ATP6AP2. We determined the RNA expression level of ATP6AP2 by RT-rtqPCi? at 72h after the transfection in U251 and U87 cell lines. We used a non-transfected control (Ctrl) to determine the baseline cell viability and the phenotype of the cells. A control scrambled siRNA (siSCR), designed to target no known gene in the cell, was used for determining the effects of siRNA delivery and for providing a baseline to compare to siRNA-treated samples. In both cell lines, the ATP6AP2 expression level is not significantly affected in the siSCR condition compared to the control. On the other hand, ATP6AP2 is strongly and significantly decreased by siATP6AP2_l (n=4) and siATP6AP2_2 (n=8) in U87 and by siATP6AP2_2 (n=4) in U251.

In our experiments, we expected that the siRNA-mediated extinction of ATP6AP2 protein could decrease tumor cells proliferation. However, double-strand RNA (dsRNA) can trigger a cellular antiviral response characterized by the activation of stress response pathways. siRNA can cause nonspecific effects through this mechanism, as for example a blocking of translation followed by the halt of cell proliferation. To monitor the antiviral response during the siRNA knockdown procedure, we analysed the expression levels of three "stress response" genes: OAS1, IFIT1 and PKR in U251 and U87. We used the transfection of a long dsRNA (500bp) as a positive control for a potent activation of the stress response. As the siATP6AP2- 2 targeting ATP6AP2 did not cause significant stress response in U251 and U87, we could rule out this nonspecific effect of the transfection procedure on cell proliferation.

Then, we assessed the protein expression by western blot. The analysis revealed a protein band at 35 kDa, corresponding to the expected full-length ATP6AP2 in both Ctrl and siSCR conditions. The absence and strong decrease of a 35 kDa band for the siATP6AP2 condition showed the knockdown efficiency in U251 (n=2) and in U87 (n=4), respectively. Thus, previous results verified that the siRNA targeting induced the knockdown of ATP6AP2 both at RNA and protein levels. The controls assessed the efficiency and the specificity of the siRNA

For example, Figure 3 teaches that siRNAs targeting ATP6AP2 efficiently induce an ATP6AP2 knock-down in U87 glioma cell lines.

Effect of the ATP6AP2 knockdown was assessed on U87 and U251 cells line in parallel. First, we observed the morphology of the cells by immunocytochemistry. siATP6AP2-targeted cells had a more rounded morphology compared to Ctrl and siSCR conditions in U87 (n=2) and U251 (n=3).

The cell population growth was evaluated by cell counting. A slight but significant decrease of cell number was observed in the siATP6AP2-2 condition 72h after the transfection of the U251 cell line (n=3). The decrease of cell number in the siATP6AP2-2 condition was stronger for the U87 cell line (n=6). This indicates that the knockdown of ATP6AP2 affects the growth capacity of a GBM cell population, more noticeably for the U87 cell line.

Then we tried to decipher the involved mechanisms by studying the cell cycle. We used Ki67 immuno labelling as a marker of proliferation, to assess the proportion of cycling cells (Gl, S, G2 and M) in each condition. Although not significant, the counting of Ki67 positive cells shows a tendency to decrease for cycling cells in siATP6AP2-2 condition for both cell lines. In addition, we performed an EdU assay to assess if the ATP6AP2 knockdown affects especially replicating cells (S-phase). For the U251 cell line, we did not observe any difference in the number of cells in phase S between controls and siATP6AP2-treated cells. For the U87 cell line, the percentage of EdU positive cells was decreased in the siATP6AP2-2 treated cells compared to the siSCR-treated cells (EdUsiATP6AP2=2% ±0,8% versus EdU_siscR=7% ±0,8%, n=l, data not shown). In parallel, we analysed the cell cycle by flow cytometry to determine the proportion of cells in each phase and to assess which phase was affected by the ATP6AP2 knockdown. For the U87 cell line, there is a trend for increase of the proportion of cells in G0/G1 phase, accompanied by a significant decrease in the S-phase, and in the G2/M-phase cell. These findings were in agreement with the Ki67 and EdU assays. siATP6AP2-2 enhanced the ratio of quiescent cells and cause a cell cycle arrest between GO and S phase and a reduced number of mitotic cells in the U87 cell line. About the U251 cell line, we cannot remark any difference in cell cycle phases between the different conditions of siR A transfection. This is in accordance with the phase-S EdU assay but not with the Ki67 assay.

These different assays indicate an effect of the ATP6AP2 knockdown by siATP6AP2-2 on the proliferative capacity of the U87 cell line. There is a blockage of cells in G0/G1 phase, inducing a decreasing number of cells in phase S and G2/M.

In summary, Figure 4 teaches that siRNAs targeting ATP6AP2 induce a decreased cell proliferation. Experiments also shown herein that ATP6AP2 knock-down in U87 glioma cell line induce an altered cell shape.

Accordingly, those experiments provide evidence that ATP6AP2 knock down by a nucleic acid molecule that down regulates the expression of prorenin receptor (PRR) in an eukaryotic cell, is an efficient active agent for reducing cell growth of glioma cell lines.

EXAMPLE 4: ATP6AP2 is expressed in GBM samples and cell lines. The expression of ATP6AP2 in brain tumours was obtained from Rembrandt (524 glioma samples). ATP6AP2 was expressed in GBM as well as in the lower grade diffuse gliomas oligodendrogliomas and astrocytomas (Figure 5A). The quartile of lower ATP6AP2 expression showed significant better survival in astrocytomas (p=0.0044) but no significant differences in GBM (p=0.23) and oligodendrogliomas (p=0.82, Figure 5B-D). According to TCGA data (349 GBM samples), the quartile of higher ATP6AP2 expression showed significant shorter survival than the lower expression quartiles (p=0.0050) in the transcriptomic proneural group of GBM. No significant differences of survival was found in patients with classical, neural or mesenchymal GBM. We checked the expression of ATP6AP2 in GBM samples with derived cell lines in our institution. RNA sequencing confirmed a stable expression of ATP6AP2 in both GBM tumor samples and derived cell lines (data not shown).

We investigated ATP6AP2 in the U87 and U251 cell lines, using reverse transcription - real-time quantitative PCR (RT-rtqPCR): both cell lines expressed ATP6AP2, with at higher level than cultured astrocytes (Figure 6B).

In addition, immunohistochemistry demonstrated the expression of ATP6AP2 by GBM samples at low level (n=3/7) or moderate level (n=3/7) while one GBM did not show detectable immunolabelling. Both lymphocytes and endothelial cells express the protein at a very low level, whereas it is highly expressed in neurons (n=7). ATP6AP2 is expressed in diffuse gliomas, patient derived cell lines and commercial GBM cell lines U87 and U251. Lower expression is associated with better survival in specific tumor types (astrocytoma, proneural GBM).

MATERIAL & METHODS

RNA sequencing. RNA were previously extracted from GBM samples of patients and from GBM derived cell lines. They were previously sequenced (internal database of Gliotex group).

Cell culture. U87 and U251 glioblastoma cell lines (ATCC®) were cultured in DMEM media, 10% Foetal Bovine Serum and 1% Penicillin/Streptomycin in a humidified incubator at 5% C02 and 37°C. Quality control of U251 cell line was done by STR DNA profiling analysis. siRNA transfection. Cells were plated the day before the transfection with 3000 cells/well (U87) or with 2000 cells/well (U251) in 96-well plates, 40000 cells/well (U87) or 30000 cells/well (U251) in 24-well plates and 15000 cells (U87) or 5000 cells (U251) for Labtek chamber slides (ThermoFisher scientific). Cells were transfected at 50-60%) confluency with siRNA (20nM final concentration) and Lipofectamine® RNAiMAX reagent diluted in Opti-MEM (ThermoFisher scientific).

- siRNA targeting ATP6AP2 (siATP6AP2_l): NM 005765.2 siRNA 390 (ThermoFisher scientific)

Sense: CCCUUUGGAGAAUGCAGUU (SEQ ID N°l) ;

Anti-Sense: AACUGCAUUCUCCAAAGGG (SEQ ID N°36)

- siRNA targeting ATP6AP2 (siATP6AP2_2): NM 005765.2 siRNA 515 (ThermoFisher scientific)

Sense: GGAAGGCAAACUCAGUGUUUU; (SEQ ID N°37)

Anti-Sense: AACACUGAGUUUGCCUUCCUU (SEQ ID N°38)

- scrambled RNA (siSCR): Stealth RNAi Negative Control, ref 12935300 (ThermoFisher scientific) - long double-strand RNA: synthetized with positive control template of MEGAscript® RNAi Kit (ThermoFisher scientific)

- siRNA targeting KIF1 1 (siKifl l): ref 1299001 , siRNA ID: HSS105842 (ThermoFisher scientific). siKIFl l transfection allowed visual control of transfection efficiency by rounded cell morphology and by

RT-rtqPCR. ARN were extracted with the Nucleospin kit (Macherey-Nagel) and dosed with NanoDrop 8000 (ThermoFisher scientific).

Reverse-transcriptase with Maxima first strand cDNA synthesis kit (ThermoFisher scientific) and we used the following program: 10min-25°C; 15min-50°C; 5min-84°C.

PCR (55 cycles), with Probe Master Mix 2x (Roche), Primers (ThermoFisher scientific)

ATP6AP2: forward: CTGAACTGCAAGTGCTACATGA; (SEQ ID N°26) APT6AP2: reverse: AACCTGCCAGCTCCAGTG; (SEQ ID N°27)

OAS 1 : forward: GGTGGAGTTCGATGTGCTG; (SEQ ID N°28)

OAS 1 : reverse: AGGTTTATAGCCGCCAGTCA; (SEQ ID N°29)

IFIT: forward: AGAACGGCTGCCTAATTTACAG; (SEQ ID N°30) IFIT reverse: GCTCCAGACTATCCTTGACCTG; (SEQ ID N°31)

PKR: forward: TGTTGGGATGGATTTGATTATG; (SEQ ID N°32) PKR reverse: GAAAAGGCACTTAGTCTTTGACCT; (SEQ ID N°33) PPIA: forward: CCTAAAGCATACGGGTCCTG; (SEQ ID N°34)

PPIA reverse: TTTCACTTTGCCAAACACCA; (SEQ ID N°35)

Probes from the Universal Probes Library (Roche): ATP6AP2 #2; OAS 1 #37; IFIT #9; PKR #54; PPIA #48. The threshold cycle number (CT) in combination with the 2^"AACT method was normalized against PPIA gene

Western Blot. Cells were lysed with RIPA buffer (ThermoFisher scientific), EDTA 0,5% and Halt™ inhibitor cocktail for 5min on ice. Then proteins were dosed with the Pierce BCA protein assay kit (ThermoFisher scientific) and loaded in denaturating conditions (2%) β-mercaptoethanol) on NuPage 4-12% Bis-Tris Gels (Novex life technologies) together with the Odyssey® One-Color Protein Molecular Weight Marker (Li-cor). Primary antibodies: ATP6AP2: 1/100, polyclonal-rabbit anti-human, HPA003156 (Sigma- Aldrich); Cyclophilin B: 1/5000, polyclonal-rabbit anti-human, SAB4200201 (Sigma- Aldrich). Secondary antibodies Odyssey anti-rabbit (ScienceTec #926-32211).

Immunocytochemistry. Cells were harvested in Labtek chamber slides, fixed with paraformaldehyde solution 3,7% in PBS during 10 min at room temperature and stained.

Primary antibodies: dilution in PBS and incubation 60min at RT

- Ki67: 1/50, monoclonal-mouse anti-human, M7240 (Dako)

- Nestin: 1/200, polyclonal-rabbit anti-human, AB5922 (Millipore)

- ATP6AP2: 1/100, polyclonal-rabbit anti-human, HPA003156 (Sigma-Aldrich)

Secondary antibodies: dilution in PBS and incubation overnight at 4°C

- Donkey anti-mouse IgG Alexa Fluor 488 conjugate, 1/200, A-21202 (ThermoFisher scientific)

- Donkey anti-rabbit IgG Cy3 conjugate, 1/200, AP182C (Millipore) Nuclei were counterstained with DAPI (Fluoromount-G with DAPI, 00-4959-52,

ThermoFisher scientific).

Immunohistochemistry. Formalin-fixed paraffin embedded tumor sections were stained with an automated Ventana benchmark XT system (Roche, Basel, Switzerland) using streptavidin-peroxidase complex with diaminobenzidin as chromogen The primary antibody was polyclonal-rabbit anti-human ATP6AP2: 1/100, HPA003156 (Sigma-Aldrich).

Automated cell counting. Cells were incubated in Nunc 96-well plates (ThermoFisher scientific) and stained with Hoechst 33342 (1/1000, Ref H3570 ThermoFisher scientific). Counting was performed with the ArrayScan™ High-Content System (ThermoFisher scientific).

Flow cytometry analysis and cell cycle analysis. Cells were fixed with cold 70 % EtOH and store at -20°C for at least 2 hours. Cells were stained with propidium iodure (50 μg/ml) in PBS with triton 0,3% and PureLink RNAse (Invitrogen, 200 μg/ml). Cell cycle was assessed by flow cytometer (BD Fortesa) and analysed with BD_Facs_Diva software. S-phase was assessed with Click- iT® Plus EdU Alexa Fluor® (Thermo scientific). Statistics analysis. Data were analyzed with GraphPad Prism 5 software. Student's t-test was performed to compare mean values between two groups. Data are presented as mean ± S.E.M.

SEQUENCE LISTING

SEQ ID Type Comment

1 RNA RNA molecule sequence targeting ATP6AP2 mRNA

2 RNA RNA molecule sequence targeting ATP6AP2 mRNA

3 RNA RNA molecule sequence targeting ATP6AP2 mRNA

4 Protein ATP6AP2 protein in view of NCBI Reference NM 005765.2

5 Protein secreted, extracellular form of the soluble part of SEQ ID N°4

6 Protein fragment derived from the transmembrane part of SEQ ID N°4

7 Protein fragment derived from the cytoplasmic part of SEQ ID N°4

8 Protein fragment of the PRR encoded by exon 4 of SEQ ID N°4

9 RNA ATP6AP2 open reading frame (NCBI Reference NM 005765.2)

10 RNA coding for secreted, extracellular form of part of SEQ ID N°4

11 RNA coding for a fragment from the transmembrane part of SEQ ID N°4

12 RNA coding for a fragment from the cytoplasmic part of SEQ ID N°4

13 RNA coding for a fragment of the PRR encoded by exon 4 of SEQ ID N°4

14 DNA forward primer

15 DNA reverse primer

16 DNA forward primer

17 DNA reverse primer

18 DNA forward primer

19 DNA reverse primer

20 DNA forward primer

21 DNA reverse primer

22 DNA forward primer

23 DNA reverse primer

24 DNA forward primer

25 DNA reverse primer

26 DNA forward primer

27 DNA reverse primer

28 DNA forward primer DNA reverse primer

DNA forward primer

DNA reverse primer

DNA forward primer

DNA reverse primer

DNA forward primer

DNA reverse primer

RNA RNA molecule sequence targeting ATP6AP2 mRNA

SEQUENCE LISTING

> SEQ ID N° 1

CCCUUUGGAGAAUGCAGUU

> SEQ ID N° 2

GGCAAACUCAGUGUUUGAA

> SEQ ID N° 3

GCUCCGUAAUCGCCUGUUU

> SEQ ID N° 4

MAVFVVLLALVAGVLGNEFSILKSPGS VVFRNGNWPIPGERIPDVAALSMGFS VKE DLSWPGLAVGNLFHRPRATVMVMVKGVNKLALPPGSVISYPLENAVPFSLDSVA NSIHSLFSEETPVVLQLAPSEERVYMVGKANSVFEDLSVTLRQLRNRLFQENSVLS SLPLNSLSR NEVDLLFLSELQVLHDISSLLSRHKHLAKDHSPDLYSLELAGLDEIG KRYGEDSEQFRDASKILVDALQKFADDMYSLYGGNAVVELVTVKSFDTSLIR TR TILEAKQAK PASPYNLAYKYNFEYSVVFNMVLWIMIALALAVIITSYNIWNMDP GYDSIIYRMTNQKIRMD

> SEQ ID N° 5

MAVFWLLALVAGVLGNEFSILKSPGSVVFRNGNWPIPGERIPDVAALSMGFSVKEDL SWPGLAVGNLFHRPRATVMVMVKGVNKLALPPGSVISYPLENAVPFSLDSVANSIHSL FSEETPVVLQLAPSEERVYMVGKANSVFEDLSVTLRQLRNRLFQENSVLSSLPLNSLSR NEVDLLFLSELQVLHDISSLLSRHKHLAKDHSPDLYSLELAGLDEIGKRYGEDSEQFR DASKILVDALQKFADDMYSLYGGNAVVELVTVKSFDTSLIRKTR > SEQ ID N° 6

TILEAKQAKNPASPYNLAYKYNFEYSWFNMVLWIMIALALAVIITSYNIWNMDPGYD SIIYRMTNQKIRMD > SEQ ID N° 7

TSYNIWNMDPGYDSIIYRMTNQKIRMD

> SEQ ID N° 8

AVPFSLDSVANSIHSLFSEETPVVLQLAPSEE

> SEQ ID N° 9

AUGGCUGUGUUUGUCGUGCUCCUGGCGUUGGUGGCGGGUGUUUUGGGGAAC GAGUUUAGUAUAUUAAAAUCACCAGGGUCUGUUGUUUUCCGAAAUGGAAAU UGGCCUAUACC AGGAGAGCGGAUCCCAGACGUGGCUGC AUUGUCC AUGGGC UUCUCUGUGAAAGAAGACCUUUCUUGGCCAGGACUCGCAGUGGGUAACCUG UUUCAUCGUCCUCGGGCUACCGUCAUGGUGAUGGUGAAGGGAGUGAACAAA CUGGCUCUACCCCCAGGCAGUGUCAUUUCGUACCCUUUGGAGAAUGCAGUU CCUUUUAGUCUUGACAGUGUUGCAAAUUCCAUUCACUCCUUAUUUUCUGAG GAAACUCCUGUUGUUUUGCAGUUGGCUCCCAGUGAGGAAAGAGUGUAUAUG GUAGGGAAGGCAAACUCAGUGUUUGAAGACCUUUCAGUCACCUUGCGCCAG CUCCGUAAUCGCCUGUUUCAAGAAAACUCUGUUCUCAGUUCACUCCCCCUCA AUUCUCUGAGUAGGAACAAUGAAGUUGACCUGCUCUUUCUUUCUGAACUGC AAGUGCUACAUGAUAUUUCAAGCUUGCUGUCUCGUCAUAAGCAUCUAGCCA AGGAUCAUUCUCCUGAUUUAUAUUCACUGGAGCUGGCAGGUUUGGAUGAAA UUGGGAAGCGUUAUGGGGAAGACUCUGAACAAUUCAGAGAUGCUUCUAAGA UCCUUGUUGACGCUCUGCAAAAGUUUGCAGAUGACAUGUACAGUCUUUAUG GUGGGAAUGCAGUGGUAGAGUUAGUCACUGUCAAGUCAUUUGACACCUCCC UCAUUAGGAAGACAAGGACUAUCCUUGAGGCAAAACAAGCGAAGAACCCAG CAAGUCCCUAUAACCUUGCAUAUAAGUAUAAUUUUGAAUAUUCCGUGGUUU UCAACAUGGUACUUUGGAUAAUGAUCGCCUUGGCCUUGGCUGUGAUUAUCA CCUCUUACAAUAUUUGGAACAUGGAUCCUGGAUAUGAUAGCAUCAUUUAUA GGAUGACAAACCAGAAGAUUCGAAUGGAUUGA > SEQ ID N° 10

AUGGCUGUGUUUGUCGUGCUCCUGGCGUUGGUGGCGGGUGUUUUGGGGAACGA GUUUAGUAUAUUAAAAUCACCAGGGUCUGUUGUUUUCCGAAAUGGAAAUUGGC CUAUACCAGGAGAGCGGAUCCCAGACGUGGCUGCAUUGUCCAUGGGCUUCUCUG UGAAAGAAGACCUUUCUUGGCCAGGACUCGCAGUGGGUAACCUGUUUCAUCGU CCUCGGGCUACCGUCAUGGUGAUGGUGAAGGGAGUGAACAAACUGGCUCUACC CCCAGGCAGUGUCAUUUCGUACCCUUUGGAGAAUGCAGUUCCUUUUAGUCUUG ACAGUGUUGCAAAUUCCAUUCACUCCUUAUUUUCUGAGGAAACUCCUGUUGUU UUGCAGUUGGCUCCCAGUGAGGAAAGAGUGUAUAUGGUAGGGAAGGCAAACUC AGUGUUUGAAGACCUUUCAGUCACCUUGCGCCAGCUCCGUAAUCGCCUGUUUCA AGAAAACUCUGUUCUCAGUUCACUCCCCCUCAAUUCUCUGAGUAGGAACAAUGA AGUUGACCUGCUCUUUCUUUCUGAACUGCAAGUGCUACAUGAUAUUUCAAGCU UGCUGUCUCGUCAUAAGCAUCUAGCCAAGGAUCAUUCUCCUGAUUUAUAUUCA CUGGAGCUGGCAGGUUUGGAUGAAAUUGGGAAGCGUUAUGGGGAAGACUCUGA ACAAUUCAGAGAUGCUUCUAAGAUCCUUGUUGACGCUCUGCAAAAGUUUGCAG AUGACAUGUACAGUCUUUAUGGUGGGAAUGCAGUGGUAGAGUUAGUCACUGUC AAGUCAUUUGACACCUCCCUCAUUAGGAAGACAAGG

> SEQ ID N° 11

ACUAUCCUUGAGGCAAAACAAGCGAAGAACCCAGCAAGUCCCUAUAACCUUGCA UAUAAGUAUAAUUUUGAAUAUUCCGUGGUUUUCAACAUGGUACUUUGGAUAAU GAUCGCCUUGGCCUUGGCUGUGAUUAUCACCUCUUACAAUAUUUGGAACAUGG AUCCUGGAUAUGAUAGCAUCAUUUAUAGGAUGACAAACCAGAAGAUUCGAAUG GAUUGA

> SEQ ID N° 12

ACCUCUUACAAUAUUUGGAACAUGGAUCCUGGAUAUGAUAGCAUCAUUUAUAG GAUGACAAACCAGAAGAUUCGAAUGGAU

> SEQ ID N° 13

GCAGUUCCUUUUAGUCUUGACAGUGUUGCAAAUUCCAUUCACUCCUUAUUUUC UGAGGAAACUCCUGUUGUUUUGCAGUUGGCUCCCAGUGAGGAA

> SEQ ID N° 14

AGCGCGTCACCTCCTCAC

> SEQ ID N° 15 AGCTTGAAATATCATGTAGCAC

> SEQ ID N° 16

CGTCCTCGGGCTACCGTCATGGTG

> SEQ ID N° 17

TTCATTGTTCCTACTCAGAGAATTGA

> SEQ ID N° 18

CGTCCTCGGGCTACCGTCATGGTG

> SEQ ID N° 19

AGCTTGAAATATCATGTAGCAC

> SEQ ID N° 20

CATGAGAAGTATGACAACAGCCT

> SEQ ID N° 21

AGTCCTTCCACGATACCAAAGT

> SEQ ID N° 22

TCTTCCCACTTTGGTTCACA

> SEQ ID N° 23

TGAGTTTGCCTTCCCTACCA

> SEQ ID N° 24

CTGGTACCCCATGGCTGTGTTTGTCGTG

> SEQ ID N° 25

GGGAATTCTCAATCCATTCGAATCTTCTG

>SEQ ID N°26

CTGAACTGCAAGTGCTACATGA; >SEQ ID N°27

AACCTGCCAGCTCCAGTG

>SEQ ID N°28

GGTGGAGTTCGATGTGCTG;

>SEQ ID N°29

AGGTTTATAGCCGCCAGTCA

>SEQ ID N°30

AGAACGGCTGCCTAATTTACAG

>SEQ ID N°31

GCTCCAGACTATCCTTGACCTG

>SEQ ID N°32

TGTTGGGATGGATTTGATTATG

>SEQ ID N°33

GAAAAGGCACTTAGTCTTTGACCT

>SEQ ID N°34

CCTAAAGCATACGGGTCCTG;

>SEQ ID N°35

TTTCACTTTGCCAAACACCA

>SEQ ID N°36

AACUGCAUUCUCCAAAGGG

>SEQ ID N°37

GGAAGGCAAACUCAGUGUUUU >SEQ ID N°38

AACACUGAGUUUGCCUUCCUU

Claims

1. A method for screening compounds for treating or preventing a tumoural and/or proliferative disorder of the central nervous system (CNS) in an individual, comprising the steps of :

c) selecting the said candidate compound if the said candidate compound inhibits the expression or biological activity of prorenin receptor (PRR) or the fragment thereof.

2. The method according to claim 1, wherein the step b) consists in determining the level of expression of said PRR or fragment thereof in said biological sample.

3. The method according to claim 1 , wherein the step b) consists in determining the concentration of said PRR or fragment thereof in said biological sample.

4. The method according to claim 1, wherein the step b) consists in determining the concentration of a nucleic acid coding for said PRR or fragment thereof in said biological sample.

5. The method according to any one of the preceding claims, comprising a step of determining the occurrence of an interaction of said candidate compound with said PRR or fragment thereof in said biological sample.

6. The method according to any one of the preceding claims, wherein said fragment of PRR comprises or consists in :

- a secreted, extracellular form of the soluble part of the PRR ; and/or - a fragment derived from the transmembrane part of the PRR; and/or

- a fragment derived from the cytoplasmic part of the PRR.

7. The method according to any one of the preceding claims, wherein said biological sample is selected from: a cerebrospinal fluid, a stem cell, a stem-cell derived neuronal cell, or an extracellular medium of any neuronal cell in primary culture.

8. The method according to any one of the preceding claims, wherein said biological sample is a neural stem cell or a glioblastoma cell line.

9. The method according to any one of the preceding claims, wherein said disorder is selected from the list consisting of glioblastomas, diffuse/infiltrating gliomas, including astrocytoma and oligodendroglioma, medulloblastoma, pineoblastoma, central primitive neuroectodermal tumour, embryonal tumour with multilayered rosettes, anaplastic ependymoma, anaplastic ganglioglioma, anaplastic pleomorphic xanthoastrocytoma. and gangliogliomas; and is preferably a glioblastoma.

10. A nucleic acid molecule that down regulates the expression of prorenin receptor (PRR) or any fragment thereof in a eukaryotic cell of an individual, for use for treating or preventing a tumoural and/or proliferative disorder of the central nervous system in said individual.

11. A nucleic acid molecule that down regulates the expression of prorenin receptor (PRR) or any fragment thereof in an eukaryotic cell of an individual according to claim 10, for use for treating or preventing a tumoural and/or proliferative disorder of the central nervous system in said individual; wherein said disorder is selected from the list consisting of gliobastomas and gangliogliomas.

12. A nucleic acid molecule that down regulates the expression of prorenin receptor (PRR) or any fragment thereof in an eukaryotic cell of an individual according to any one of claims 10 or 11, for use for treating or preventing a tumoural and/or proliferative disorder of the central nervous system in said individual; wherein said nucleic acid molecule is selected from the group consisting of: ribozymes, antisense nucleic acids, short interfering nucleic acids (siNA), short-interfering RNAs (siRNAs), micro-RNAs (miRNA), short hairpin RNAs (shRNA), short interfering oligonucleotide, short interfering substituted oligonucleotide, short interfering modified oligonucleotide.

13. A nucleic acid molecule that down regulates the expression of prorenin receptor (PRR) or any fragment thereof in an eukaryotic cell of an individual according to claim 12, for use for treating or preventing a tumoural and/or proliferative disorder of the central nervous system in said individual; wherein said nucleic acid molecule is a siRNA, comprising at least one sequence selected from the group consisting of SEQ ID N°l to 3.

14. A nucleic acid molecule that down regulates the expression of prorenin receptor (PRR) or any fragment thereof in a eukaryotic cell of an individual; comprising at least one sequence selected from the group consisting of SEQ ID N°l and 2.

15. A pharmaceutical composition comprising a nucleic acid molecule that down regulates the expression of prorenin receptor (PRR) or any fragment thereof in an eukaryotic cell of an individual according to any one of claims 10 to 14, for use for treating or preventing a tumoural and/or proliferative disorder of the central nervous system in an individual.