Isolated or artificial nucleotides for use in treating neurodegenerative diseases

Abstract

The present disclosure relates to an isolated or artificial nucleotide sequence encoding GTPase-activating protein-binding protein 1 (G3BP1) for use in medicine, preferably in the treatment of polyglutamine diseases. Furthermore, the present invention also relates to a vector comprising such a sequence, a host cell comprising such a vector, the protein G3BP1, or compositions thereof, for use in medicine, preferably in the treatment of polyglutamine diseases.

Description

[Technical field]

[0001] The present disclosure relates to isolated or artificial nucleotide sequences encoding GTPase-activating protein-binding protein 1 (G3BP1) for use in medicine, preferably in the treatment of polyglutamine diseases. Furthermore, the present invention also relates to such sequences, host cells comprising such vectors, vectors comprising the protein G3BP1, or compositions thereof, for use in medicine, preferably in the treatment of polyglutamine diseases. [Background technology]

[0002] Polyglutamine (PolyQ) diseases are a group of inherited neurodegenerative disorders that include Huntington's disease (HD), spinal-bulbar muscular atrophy (SBMA), dentatorubral-pallidoluysian atrophy (DRPLA), and several spinocerebellar ataxias (SCA1, 2, 3, 6, 7, 17). These diseases are characterized by abnormal expansion of the trinucleotide CAG in the coding region of each disease-related gene, which encodes an expanded polyglutamine tract in the respective protein. A central feature of these diseases is the aggregation of mutant proteins, which promotes abnormal interactions with other proteins and mRNAs, impairing the functioning of several cellular pathways and organelles. 1 Nevertheless, the complete picture of the molecular events that lead to selective neurodegeneration in certain brain regions is still not fully understood. Moreover, to date, there is no treatment that can halt or slow the progression of the disease, which leads to the premature death of PolyQ disease patients.

[0003] SCA2 and SCA3 (or Machado-Joseph disease - MJD) are the two most common spinocerebellar ataxias, both characterized by a neurodegenerative profile that primarily affects the cerebellum and brainstem. SCA2 is caused by abnormal mutations in the ATXN2 gene above CAG repeats 31-33, resulting in an over-expanded ataxin-2 protein2. SCA3 is caused by abnormal mutations in the ATXN3 gene above CAG repeats 44-45, resulting in an over-expanded ataxin-3 protein2. 3,4 Expand. Both mutant ataxin-2 and ataxin-3 are prone to aggregation and form large inclusions that can sequester other proteins. Large inclusions are often reported as a hallmark of disease, but whether they directly translate to toxicity remains controversial. 5-8 .

[0004] Pathological aggregation of PolyQ proteins and abnormal interactions involving PolyQ proteins can significantly alter cellular stress response pathways. 9,10 To cope with stress, cells display several mechanisms that promote survival, including the assembly of stress granules (SGs), transiently formed foci that act to triage and regulate RNA during stress. 11 In recent years, SG dysregulation has been suggested to underlie the pathogenesis of several diseases, including neurodegenerative disorders. 12 SGs are dynamic structures that are mainly composed of RNA-binding proteins (RBPs) but can have different compositions depending on the type of stress and the type of cell. One of these components, which is also a marker and core nucleator of SGs, is the GTPase-activating protein-binding protein 1 (G3BP1). 13,14 G3BP1 plays an important role in mRNA stabilization, degradation, and splicing regulation 15,16,17Structurally, G3BP1 has at least two important domains, an RNA recognition domain (RRM) and a nuclear transport factor 2-like domain (NTF2-like). The former is important for the mRNA-binding ability of G3BP1, whereas the latter is involved in the pore complex. 15 Involved in nuclear transport of proteins via 15 Furthermore, phosphorylation of G3BP1 at its Ser-149 residue has been noted to be important in SG assembly, although recent studies do not support his hypothesis. Although all these domains and catalytic sites are important for the function of G3BP1, the role of each of them in the specific steps of RNA metabolism remains to be elucidated. 13,14 .

[0005] Despite extensive efforts developed over the past few years, the pathogenesis of polyQ diseases is not fully understood and no therapeutic options exist that slow or halt disease progression. Therefore, it is essential to identify new molecular targets involved in disease pathogenesis and develop new therapeutic strategies for this group of diseases.

[0006] These facts are disclosed to explain the technical problem addressed by the present disclosure. Summary of the Invention

[0007] In this study, we investigated the involvement of G3BP1, a SG component in the pathogenesis of SCA2 and SCA3, and its suitability as a therapeutic target. We observed that overexpression of G3BP1 led to a significant decrease in the number of cells with aggregates and the levels of ataxin-2 and ataxin-3 proteins. The NTF2-like domain and the Ser149 residue appear to be important for this mechanism of action of G3BP1. Furthermore, we found that G3BP1 levels were decreased in samples from SCA2 and SCA3 patients. Importantly, knockdown of G3BP1 levels increased the number of aggregates in lentiviral mouse models of SCA2 and SCA3, highlighting the important functional role of this protein in the context of SCA2 and SCA3. Conversely, re-establishment of G3BP1 levels in lentiviral mouse models of SCA2 and SCA3 reduced the neuropathological abnormalities associated with the expression of mutant ataxin-2 or mutant ataxin-3, respectively. In the same line, G3BP1 expression was able to significantly reduce behavioral and neuropathological deficits in a transgenic mouse model. Overall, we have surprisingly identified G3BP1 as a relevant target for PolyQ diseases, namely SCA2 and SCA3, and disclosed a therapeutic strategy for such diseases.

[0008] The present inventors have shown that there is a decrease in G3BP1 levels in patients with SCA2 and SCA3 diseases. Based on this discovery, the present inventors have successfully investigated the possibility of modulating G3BP1 expression as a therapeutic strategy against SCA2 and SCA3 by using a vector encoding a nucleic acid that expresses G3BP1 in target cells.

[0009] It is therefore an object of the present invention to provide an isolated or artificial nucleotide sequence encoding GTPase-activating protein-binding protein 1 (G3BP1) for use in medicine (medical) or veterinary medicine (veterinary), preferably in the treatment of polyglutamine diseases. Furthermore, the present invention also relates to such a sequence, a host cell comprising such a vector, a vector comprising the protein G3BP1 or compositions thereof, for use in medicine or veterinary medicine, preferably in the treatment of polyglutamine diseases.

[0010] According to one embodiment, the G3BP1 protein is the protein identified by NCBI sequence reference: NP_005745.1, which is encoded by a nucleic acid sequence identified by NCBI sequence reference GI:10146 (GeneBank accession: NM_005754.3).

[0011] One aspect of the present disclosure relates to an isolated or artificial nucleotide sequence encoding the protein G3BP1 for use in pharmaceutical or veterinary medicine, wherein the sequence is at least 95% identical to a sequence selected from the list consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and mixtures thereof.

[0012] In one embodiment, the isolated or artificial nucleotide sequence for use in pharmaceutical or veterinary medicine is identical to a sequence selected from the list consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and mixtures thereof.

[0013] According to one embodiment, the isolated or artificial nucleotide sequences may be used to treat disorders of the central and peripheral nervous system.

[0014] In another embodiment, the isolated or artificial nucleotide sequence may be used to treat a neurodegenerative disease.

[0015] In another embodiment, the isolated or artificial nucleotide sequence may be used to treat movement disorders, ie deficits in balance, motor coordination and / or motor performance.

[0016] In another embodiment, the isolated or artificial nucleotide sequence may be used to treat a polyglutamine disease.

[0017] In another embodiment, the isolated or artificial nucleotide sequence may be used for the treatment of a polyglutamine disease, which is positively affected by the control of protein aggregation, wherein said control of protein aggregation is caused by an expansion in a polyglutamine segment of the affected protein.

[0018] In another embodiment, the isolated or artificial nucleotide sequence may be used in the treatment of a polyglutamine disease, wherein the disease is selected from the group consisting of Huntington's disease (HD), spinal-bulbar muscular atrophy (SBMA), dentatorubral-pallidoluysian atrophy (DRPLA), and polyglutamine repeat spinocerebellar ataxia.

[0019] In another embodiment, the isolated or artificial nucleotide sequence may be used to treat a polyglutamine repeat spinocerebellar ataxia, wherein the polyglutamine repeat spinocerebellar ataxia is selected from the group consisting of spinocerebellar ataxia type 1 (SCA1), spinocerebellar ataxia type 2 (SCA2), spinocerebellar ataxia type 3 (SCA3), spinocerebellar ataxia type 6 (SCA6), spinocerebellar ataxia type 7 (SCA7) and spinocerebellar ataxia type 17 (SCA17).

[0020] In another embodiment, the isolated or artificial nucleotides may be administered directly into the patient's brain or into the patient's spinal cord.

[0021] In another embodiment, the isolated or artificial nucleotide may be administered by intravascular, intravenous, intranasal, intracerebroventricular or intrathecal injection.

[0022] Another aspect of the present disclosure relates to a vector or construct comprising an isolated or artificial nucleotide sequence as described above.

[0023] In one embodiment, the vector is selected from the group of adenovirus, lentivirus, retrovirus, herpesvirus and adeno-associated virus (AAV) vectors.

[0024] In another embodiment, the vector is a lentiviral vector.

[0025] Another aspect of the present disclosure relates to a host cell comprising the above-described vector for use in pharmaceutical or veterinary medicine.

[0026] Another aspect of the present disclosure relates to a protein G3BP1 encoded by an isolated or artificial nucleotide sequence for use in pharmaceutical or veterinary medicine, wherein the sequence is at least 95% identical to a sequence selected from the list consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and mixtures thereof.

[0027] Another aspect of the present disclosure relates to a pharmaceutical composition for use in human or veterinary medicine comprising a therapeutically effective amount of the above-mentioned isolated or artificial nucleotide sequence, or the above-mentioned vector, or the above-mentioned host cell, or the above-mentioned protein, or a combination thereof.

[0028] Another aspect of the present disclosure relates to a kit for use in pharmaceutical or veterinary medicine comprising the above-mentioned isolated or synthetic nucleotide sequence, or the above-mentioned vector, the above-mentioned host cell, or the above-mentioned protein, or a combination thereof.

[0029] The following figures provide preferred embodiments to illustrate the disclosure and should not be considered as limiting the scope of the invention. [Brief description of the drawings]

[0030] [Figure 1] Sodium arsenite-mediated stress granule assembly does not alter the number and protein levels of ATXN2 and ATXN3 aggregates. a, Representative confocal microscopy images from Neuro2a cells expressing pathological and non-pathological forms of ATXN2 fused to an eGFP tag (top panel), treated with sodium arsenite to induce stress granule (SG) assembly, and stained with an antibody against the SG marker PABP (middle panel). b, Representative western blots of Neuro2a lysates expressing pathological and non-pathological forms of ATXN2 and treated with sodium arsenite to induce SG assembly. Western blots were labeled with ATXN2, phospho-eIF2α, and β-tubulin antibodies. c, The number of cells with ATXN2 aggregates did not change significantly upon SG assembly (n=3 independent experiments). d, The levels of non-pathological ATXN2 (ATXN2WT) protein did not change significantly upon SG assembly. e, The levels of pathological ATXN2 (ATXN2MUT) protein did not change significantly upon SG assembly. (n=5 independent experiments). f, Representative confocal microscopy images from Neuro2a cells expressing pathological and non-pathological forms of ATXN3 fused with eGFP tag (upper panel), treated with sodium arsenite to induce SG assembly, and stained with an antibody against PABP1. g, Representative western blots of Neuro2a lysates expressing pathological and non-pathological forms of ATXN3 and Neuro2a lysates treated with sodium arsenite to induce SG assembly. Western blots were labeled with ATXN3, phospho-eIF2α, and β-tubulin antibodies. h, The number of cells with ATXN3 aggregates did not change significantly upon SG assembly (n=3 independent experiments). i, Non-pathological ATXN3 (ATXN3WT) protein levels did not change significantly upon SG assembly. j, Pathological ATXN3 (ATXN3MUT) protein levels did not change significantly upon SG assembly (n=5 independent experiments). Values ​​are expressed as mean ± standard error of the mean (SEM). Scale: 10 μm. [Diagram 2] Expression of G3BP1 reduces the number of cells with ATXN2MUT and ATXN3MUT aggregates. a, Representative confocal microscopy images depicting Neuroa2 cells expressing ATX2MUT in three different experimental conditions. b, Representative confocal microscopy images depicting Neuroa2 cells expressing ATX3MUT in three different experimental conditions. c, The number of cells with ATX2MUT aggregates per 100 transfected cells was significantly reduced upon G3BP1 expression compared to both control conditions. d, The number of cells with ATX3MUT aggregates per 100 transfected cells was significantly reduced upon G3BP1 expression compared to both control conditions. (n=3 independent experiments, *P<0.05; one-way ANOVA followed by post hoc Bonferroni multiple comparison test). Values ​​are expressed as mean ± SEM. Scale: 10 μm. e, Representative Western blots of Neuro2a lysates expressing pathological and non-pathological forms of ATXN2 co-transfected with lacZ or G3BP1. f, Representative Western blots of Neuro2a lysates expressing pathological and non-pathological forms of ATXN2 co-transfected with lacZ or G3BP1. g, ATXN2WT protein levels are significantly reduced upon G3BP1 expression compared to control cells co-expressing lacZ. h, ATXN2MUT protein levels are significantly reduced upon G3BP1 expression compared to control cells co-expressing lacZ. i, ATXN3WT protein levels are significantly reduced upon G3BP1 expression compared to control cells co-expressing lacZ. j, ATXN3MUT protein levels are significantly reduced upon G3BP1 expression compared to control cells co-expressing lacZ. (n=5 independent experiments; *p<0.05; **p<0.01; Student's t test). [Diagram 3]The NFT2-like domain of G3BP1 is important in the modulation of aggregation and protein levels of ATXN2MUT and ATXN3MUT. a, Schematic representation of G3BP1 structural domain and respective constructs with deleted NFT2 domain and deleted RRM domain. Δ: deletion. NTF2: nuclear transport factor 2 domain. Ser: serine. PxxP: proline-rich region. RRM: RNA recognition motif. RGG box: arginine- and glycine-rich box. b, Neuro2a cells were transfected with either full-length G3BP1, G3BP1-ΔRRM or G3BP1-ΔNTF. Protein lysates were analyzed by Western blot showing the expression of G3BP1 truncated forms with different molecular weights. c, Representative confocal microscopy images showing Neuroa2 cells expressing ATXN2MUT and lacZ or G3BP1 or G3BP1-ΔNTF2 or G3BP1-ΔRRM. Expression of ATXN2MUT leads to the formation of aggregates (arrows). d, Representative confocal microscopy images depicting Neuroa2 cells expressing ATXN3MUT and lacZ or G3BP1 or G3BP1‐ΔNTF2 or G3BP1‐ΔRRM. Expression of ATXN3MUT leads to the formation of aggregates (arrows). Scale: 10 μm. e, The number of cells with aggregates of ATX2MUT per 100 transfected cells with G3BP1‐ΔRRM was significantly decreased compared to the lacZ control condition and increased compared to the full-length G3BP1 condition. Expression of G3BP1‐ΔNTF2 led to a significant increase in the number of cells with aggregates compared to all other experimental conditions. (n=4 independent experiments; ##P<0.01 vs. ATXN2MUT+lacZ; ****P<0.0001 vs. ATXN2MUT+G3BP1; ++++P<0.0001 vs. ATXN2MUT+G3BP1‐ΔRRM; one-way ANOVA followed by post hoc Bonferron multiple comparisons test).f, The number of cells with aggregates of ATX3MUT per 100 transfected cells with G3BP1-ΔRRM was significantly decreased compared to the lacZ control condition and increased compared to the full-length G3BP1 condition. Expression of G3BP1-ΔNTF2 significantly increases the number of cells with aggregates compared to ATXN3MUT+G3BP1 and ATXN3MUT+G3BP1-ΔRRM. (n=4 independent experiments; ##P<0.01 vs. ATXN3MUT+lacZ; ****P<0.0001 vs. ATXN3MUT+G3BP1; ++P<0.01 vs. ATXN3MUT+G3BP1-ΔRRM; one-way ANOVA followed by post-hoc Bonferroni multiple comparison test). g, Representative western blots of Neuro2a lysates expressing ATXN2MUT cotransfected with lacZ or G3BP1-ΔRRM or G3BP1-ΔNTF2. h, ATXN2MUT protein levels (concentrations) are significantly decreased by G3BP1-ΔRRM expression compared to other experimental conditions, whereas G3BP1-ΔNTF2 significantly increases ATXN2MUT protein levels compared to other conditions (n=4 independent experiments; **P<0.01 for ATXN2MUT+lacZ; ####P<0.0001 for ATXN2MUT+G3BP1-ΔRRM; one-way ANOVA followed by post hoc Bonferroni multiple comparison test). i, Representative western blots of Neuro2a lysates expressing ATN3MUT cotransfected with lacZ or G3BP1-ΔRRM or G3BP1-ΔNTF2. j, ATXN3MUT protein levels are significantly decreased by G3BP1-ΔRRM expression compared to other experimental conditions, whereas expression of G3BP1-ΔNTF2 results in a significant increase in ATXN3MUT protein levels compared to ATXN3MUT+G3BP1-ΔRRM. (n=4 independent experiments; *P<0.05 vs. ATXN3MUT+lacZ; **P<0.01 vs. ATXN3MUT+lacZ; ###P<0.001 vs. ATXN3MUT+G3BP1-ΔRRM; one-way ANOVA followed by post-hoc Bonferroni multiple comparison test). Values ​​are expressed as mean ± SEM. [Figure 4]The Ser149 phosphorylation site is important for the action of G3BP1 on ataxin-2 and ataxin-3 mutant proteins. a, Representative confocal microscopy images showing Neuroa2 cells expressing ATXN2MUT and wild-type G3BP1 or GB3BP1(S149A) or GB3BP1(S149D). In cells expressing wild-type G3BP1 or the phosphomimetic G3BP1(S149D), aggregates of ATXN2MUT are absent (white arrows), in contrast to cells expressing phospho-dead G3BP1(Ser149A), where aggregates of ATXN2MUT are observed (white arrowheads). b, Representative confocal microscopy images showing Neuroa2 cells expressing ATXN3MUT and wild-type G3BP1 or GB3BP1(S149A) or GB3BP1(S149D). In cells expressing wild-type G3BP1 or phospho-mimetic G3BP1(S149D), aggregates of ATXN3MUT (white arrows) are absent, in contrast to cells expressing phosphorylation-dead G3BP1(Ser149A). Scale: 20 μm. c, Representative Western blot of Neuro2a lysates expressing ATXN3MUT, co-transfected with wild-type G3BP1 or G3BP1(S149A) or G3BP1(S149D). d, Representative Western blot of Neuro2a lysates expressing ATXN3MUT, co-transfected with wild-type G3BP1 or G3BP1(S149A) or G3BP1(S149D). e, ATXN2MUT protein levels are significantly increased in cells expressing phosphorylated death G3BP1 (Ser149A) compared to ATX2MUT+G3BP1 (n=3 independent experiments; *P<0.05; one-way ANOVA followed by post-hoc Bonferroni multiple comparison test). f, No significant changes in ATX2MUT mRNA levels were observed between all experimental conditions. g, ATXN2MUT protein levels were significantly increased in cells expressing phosphorylated death G3BP1 (Ser149A) compared to the other two conditions, whereas cells expressing phosphorylated mimic G3BP1 (Ser149D) were significantly decreased compared to the other two conditions.(n=3 independent experiments; *P<0.05 vs. ATXN3MUT+G3BP1; ##P<0.01 vs. ATXN3MUT+G3BP1(Ser149D); one-way ANOVA followed by post-hoc Bonferroni multiple comparison test.) Values ​​are expressed as mean ± SEM. [Diagram 5]G3BP1 mRNA and protein levels are reduced in SCA2 and SCA3, and its silencing in mouse brain increases aggregation. a, Representative Western blots of protein lysates from fibroblasts from SCA2 patients and healthy controls. b, Representative Western blots of protein lysates from fibroblasts from SCA3 patients and healthy controls. c, d, G3BP1 protein and mRNA levels are significantly reduced in SCA2 compared to controls. e, f, G3BP1 protein and mRNA levels are significantly reduced in SCA3 compared to controls. (healthy controls n=3; SCA2 n=2; SCA3 n=5) g, Representative Western blots of protein lysates of cerebellum from transgenic SCA3 mouse models. h, i, G3BP1 protein and mRNA levels are significantly reduced in SCA3 transgenic mice compared to C57BL / 6 wild-type animals (n=3–5). j, Schematic diagram of injection site for SCA2 model. Briefly, lentiviral vectors encoding shRNA-scrambled and ATXN2MUT were co-injected into one hemisphere of the striatum, and shRNA targeting G3BP1 and ATXN2MUT were co-injected into the contralateral hemisphere. k, Four weeks after injection, animals were euthanized and brain sections labeled with ataxin-2 were prepared to highlight the presence of pathological aggregates. l, The average number of ATXN2MUT aggregates is significantly increased upon shG3BP1 expression compared to control hemispheres. m, Schematic diagram of injection site in SCA2 model. Briefly, lentiviral vectors encoding shRNA-scrambled3ere and ATXN3MUT were co-injected into one hemisphere of the striatum, and shRNA targeting G3BP1 and ATXN2MUT were co-injected into the contralateral hemisphere. n, Four weeks after injection, animals were euthanized and brain sections labeled with ataxin-3 were prepared to highlight the presence of pathological aggregates. o, The mean number of ATXN3MUT aggregates is significantly increased upon shG3BP1 expression compared to control hemispheres (*P<0.05; **P<0.01; ***P<0.001; Student's t test). Values ​​are expressed as mean ± SEM. [Figure 6]Expression of G3BP1 reduces aggregate numbers and loss of neuronal markers in lentiviral mouse models of SCA2 and SCA3. a) Mice were stereotactically injected into the striatum with lentiviral particles encoding ATXN2 or ATXN3 mutants or co-injected with lentiviral particles encoding mutants and G3BP1. a) Schematic of lentiviral vectors injected into a mouse model of SCA2 and injection sites. Animals were injected bilaterally and euthanized 12 weeks after injection and tissues were harvested. b) Schematic of lentiviral vectors injected into a mouse model of SCA3 and injection sites. Animals were injected bilaterally and euthanized 4 weeks after injection and tissues were harvested. c) Brain sections from a lentiviral mouse model of SCA2 were analyzed by immunohistochemistry with ataxin-2 and DARP-32 antibodies. The images show aggregates of ATXN2MUT (black arrowheads; scale: 20 μm) and loss of staining of the neuronal marker DARPP-32 (black line) (scale: 200 μm). d, G3BP1-expressing hemispheres showed a reduction in the number of ATXN2MUT aggregates compared to control hemispheres (n=5; *** p<0.0001; Student's t-test). e, G3BP1 expression rescues neuronal marker loss compared to the contralateral hemisphere expressing only ATXN2MUT (n=5; *** p<0.0001; Student's t-test). f, Representative images of immunohistochemical brain sections from a lentiviral mouse model of SCA3. The figures show ubiquitinated ATXN3MUT aggregates (scotoma; scale: 20 μm) and loss of staining of the neuronal marker DARPP-32 (scale: 200 μm). g, Expression of G3BP1 led to a significant decrease in the number of ubiquitinated ATXN3MUT aggregates compared to control conditions (n=7; *** p<0.0001; Student's t-test). h, Expression of G3BP1 rescues neuronal marker loss compared to controls (n=7; *** p<0.0001; Student's t-test). Values ​​are expressed as mean ± SEM. [Figure 7]Overexpression of lentiviral vectors encoding G3BP1 in wild-type mouse brains did not result in loss of neuronal markers or inflammation. Mice aged 8–12 weeks were stereotaxically injected (bilaterally) into the striatum with PBS or lentiviral particles encoding human G3BP1 and euthanized for tissue collection 4 weeks after injection. a, Schematic of injection sites in the striatum. b, Immunohistochemical image analysis of DARPP-32 depletion volume (black dashed line; upper panel; scale: 200 μm) and G3BP1 (lower panel; scale: 50 μm) labeling in brain sections of mice injected with PBS and in the contralateral hemisphere injected with lentiviral particles encoding G3BP1. c, The total area of ​​DARPP-32 depletion in mouse brain sections was reduced by injection of lentiviral particles encoding G3BP1 when compared with injection of PBS (n=4; *P<0.05; Student's t-test). e, Quantification of GFAP immunoreactivity did not detect significant differences between the two hemispheres. Scale: 200 μm. Values ​​are expressed as mean ± standard error. [Figure 8]Expression of G3BP1 reduces motor deficits and neuropathological abnormalities in a SCA3 transgenic mouse model. Transgenic mice expressing a truncated form of the ataxin-3 protein containing 69 glutamines were stereotactically injected into the cerebellum with lentiviral particles encoding GFP (control group) or G3BP1 (treated group). Four-week-old mice were first tested 1–2 days before injection, repeatedly tested every 3 weeks until 9 weeks after injection, and euthanized 10 weeks after surgery. a–c, Representative plots of the motor performance of mice at 9 weeks after injection. a, Mice injected with G3BP1 had significantly improved motor performance (assessed by rotarod test) as they remained more time on the rotating rod compared to control mice treated with GFP or non-injected mice. b, Mice injected with G3BP1 had significantly reduced the time required to traverse the water-filled tank and reach the platform compared to control mice treated with GFP or non-injected animals. c, Footprint analysis showed that mice injected with G3BP1 had improved overlap measurements compared to controls treated with GFP or non-injected animals. d, Representative images of immunohistochemical brain sections from cerebella of mice injected with lentiviral particles encoding GFP (control) or G3BP1. Top panel: ataxin-3 aggregates assessed by HA-tag immunoreactivity (arrowheads; scale: 50 μm and 200 μm). Bottom panel: Purkinje cells assessed by calbindin immunoreactivity (scale: 100 μm and 200 μm). e, G3BP1 expression significantly reduced the number of HA-ataxin-3 aggregates (arrowheads) compared to GFP-injected or non-injected control mice. f, G3BP1 expression significantly preserved the number of Purkinje cells (red) within lobe IX compared to non-injected and GFP-injected controls. (n = 6–7; *p < 0.05; one-way ANOVA followed by post-hoc Bonferroni multiple comparison test). Values ​​are expressed as mean ± SEM. [Figure 9]Sodium arsenite induces stress granule formation in Neuro2a cells and reduces global protein synthesis. (A) Representative confocal microscopy images of Neuro2a untreated and treated with sodium arsenite 1 h prior to fixation, showing SG marker PABP immunolabeling. Condensate foci PABP-positive SGs can be observed in treated cells. (B) Representative western blot of protein lysates from Neuro2a cells after sunset assay. Cells were transfected with G3BP1 or treated with sodium arsenite to induce stress granule formation. As a protein synthesis inhibition control, cells were treated with cycloheximide (CHX) and membranes were probed with puromycin antibody. (C) SG induction by sodium arsenite reduces global protein expression. (D) G3BP1-transfected cells also have significantly reduced global protein synthesis compared to lacZ conditions. Furthermore, total protein synthesis inhibition upon G3BP1 expression showed no difference compared to treatment with cycloheximide, a known protein synthesis inhibitor (n=4; **p<0.001; ****p<0.00001; one-way ANOVA followed by post-hoc Bonferroni multiple comparison test). Values ​​are expressed as mean ± SEM. Scale: 10 μm. [Figure 10] Expression of G3BP1 and lacZ plasmids in Neuro2a cells. a, Representative Western blots of protein lysates from Neuro2a cells transfected with G3BP1 or lacZ 48 h post-transfection. Western blots were labeled with G3BP1, β-gal and β-actin antibodies. b, Representative confocal images from Neuro2a cells transfected with lacZ and immunolabeled with β-gal (white arrows) and Neuro2a cells transfected with G3BP1 and immunolabeled with G3BP1. Nuclei were stained with DAPI (blue). Scale bar: 10 μm. [Figure 11]Expression of G3BP1 does not alter the levels of endogenous mouse Ataxin-2 and Ataxin-3 proteins. Neuro2a cells were co-transfected with ATXN2MUT and G3BP1 or ATXN3MUT and G3BP1. Endogenous levels of mouse Ataxin-2 and Ataxin-3 proteins were assessed. G3BP1 overexpression did not alter the endogenous levels of both (A) endogenous Ataxin-2 and (B) endogenous Ataxin-3 compared to control conditions (n ​​= 4–5 independent experiments; Student's t test). Values ​​are expressed as mean ± SEM. [Figure 12] Overexpression of G3BP1 does not change the expression levels of GFP. Neuro2a cells were transfected with GFP or cotransfected with GFP and G3BP1. (A) Representative blot of Neuro2a protein lysates immunoblotted with anti-GFP antibody. (B) GFP expression was not altered by G3BP1 expression (n=4 independent experiments; Student's t test). Values ​​are expressed as mean ± SEM. [Figure 13] When stress granules are pharmacologically induced with sodium arsenite, G3BP1 colocalizes with PABP. Neuro2a cells were transfected with G3BP1 and treated with sodium arsenite 1 h before adhesion. Representative immunocytochemistry images from Neuro2a cells showing G3BP1 labeling. Immunolabeling of the stress granule marker protein PABP is shown in red. In Neuro2a cells transfected with G3BP1, no colocalization with PABP was observed in the absence of any treatment. In Neuro2a cells transfected with G3BP1 and SG assemblies, when pharmacologically induced with sodium arsenite treatment, PABP colocalizes with G3BP1 (yellow). Scale: 10 μm. [Figure 14]G3BP1 aggregates (assembles) into PABP-positive stress granules upon sodium arsenite-induced stress. Control fibroblasts from healthy individuals and SCA2 and SCA3 patients were treated with sodium arsenite for 1 h before fixation. Cells were immunolabeled for G3BP1 (green) and PABP (red) and screened for colocalization of both proteins using confocal microscopy. Representative images show that G3BP1 aggregates into SGs upon sodium arsenite-induced stress, highlighted by its colocalization with PABP, a marker for SGs. Nuclei were stained with DAPI (blue). Scale bar: 50 μm [Figure 15] Site-directed mutagenesis of the serine-149 phosphorylation site. (A) Schematic of the G3BP1 gene structure with a mutagenesis site that changed the serine to an alanine, creating a phospho-dead construct at the 149aa site, G3BP1(S149A). (B) Schematic of the G3BP1 gene structure with a mutagenesis site that changed the serine to an aminotransferase, creating a phospho-mimetic construct at the 149aa site, G3BP1(S149D). (C) Representative image of the G3BP1 (SEQ ID NO: 1) coding region near the serine-149 site. The top histogram shows the G3BP1 sequence before locus-directed mutagenesis. The middle histogram shows G3BP1 after site-directed mutagenesis targeting serine-149->alanine149. The first thymine (T) nucleotide in the triplet TCT (coding for serine) was replaced by a guanine originating from the triplet GCT (coding for alanine). The histogram below shows G3BP1 after site-directed mutagenesis targeting Serine-149->Aspartic acid 149. The thymine (T) and cytosine (C) nucleotides in the triplet TCT (coding for serine) were replaced by a guanine and an adenine originating from the triplet GAT (coding for aspartic acid). [Figure 16]Expression of G3BP1 reduces the levels of mutant ATXN2 and ATXN3 proteins. (A) The level of ATXN2MUT mRNA is reduced upon expression of G3BP1 compared to control conditions (ATXN2MUT+lacZ). (B) In the same line, the level of ATXN3MUT mRNA is reduced upon expression of G3BP1 compared to control conditions (ATXN3MUT+lacZ). (n=4 independent experiments; *P<0.05; ***P<0.001; Student's t test). Values ​​are expressed as mean ± SEM. [Figure 17] Immunostaining for G3BP1 is reduced in postmortem brain samples from SCA2 patients. Representative images of immunohistochemistry in human brain samples. Postmortem human brain biopsies from healthy individuals and SCA2 patients were immunohistologically stained for G3BP1. Upper panel: G3BP1 immunodetection from the striatum. Lower panel: G3BP1 immunodetection from the cerebellum. G3BP1 staining in the cerebellum and striatum was abolished in SCA2 patients when compared to healthy individuals. Samples from two diagnosed SCA2 patients and three healthy controls were analyzed. Scale: 100 μm and 400 μm. [Figure 18] There is reduced immunostaining for G3BP1 in Purkinje cells of a transgenic SCA3 mouse model. Representative confocal images of immunohistochemistry for G3BP1 and calbindin in wild-type C57BL / 6 mice and transgenic SCA3 mice expressing a mutant ataxin-3 with 69 glutamines in Purkinje cells of the cerebellum. In these cells, it is possible to observe reduced immunostaining for G3BP1 in the transgenic animals compared to wild-type mice. Scale: 10 μm. [Figure 19]shRNA targeting G3BP1 significantly reduces its levels. (A) Representative western blot from Neuro2a lysates transfected with validated shRNA targeting mouse G3BP1 and control shRNA scramble. (B) shG3BP1 results in a significant reduction in the levels of mouse endogenous G3BP1 protein compared to control. (C) shG3BP1 results in a significant reduction in the levels of mouse endogenous G3BP1 mRNA compared to control conditions. (n=2-3 independent experiments; *P<0.05; **P<0.01; Student's t-test). Values ​​are expressed as mean ± standard error. [Figure 20] Expression of G3BP1 in the striatum regulates the levels of ATXN3MUT in a lentiviral mouse model of SCA3. (A) Representative Western blots of protein lysates from striatal punches from mice injected with lentiviral particles encoding ATXN2MUT in one striatal hemisphere, and co-injected with lentiviral particles encoding ATXN2MUT and G3BP1 in the contralateral hemisphere (4 weeks post-injection). (B) No significant changes in soluble levels of ATX2MUT between striatal hemispheres were observed. (C) ATXN2MUT mRNA levels were similar in both hemispheres. (D) Representative Western blots of protein lysates from striatal punches from mice injected with lentiviral particles encoding ATXN3MUT in one striatal hemisphere, and co-injected with lentiviral particles encoding ATXN3MUT and G3BP1 in the contralateral hemisphere (4 weeks post-injection). (E) ATX3MUT levels were reduced in the G3BP1-expressing compared to control hemispheres. (F) ATXN3MUT mRNA levels were similar in both hemispheres (SCA2 n=3 and SCA3 n=4, Student's t test). Values ​​are expressed as mean ± SEM. [Figure 21]Non-transduced cerebellar lobes of SCA3 transgenic mice injected with G3BP1 did not show any neuropathological reduction. SCA3 transgenic mice expressing a truncated form of ataxin-3 protein containing 69 glutamines were stereotactically injected into the cerebellum with lentiviral particles encoding G3BP1 (treated group) or with lentiviral particles encoding GFP (control group). (A) Immunohistochemical analysis of brain sections of mice cerebellum treated with lentiviral particles encoding GFP (control) or with lentiviral particles encoding G3BP1. Shown in the upper panel: ataxin-3 aggregates assessed by HA tag immunoreactivity; shown in the lower panel: cerebellar Purkinje cells assessed by calbindin immunoreactivity. Scale: 200 μm. Non-transduced lobes (VII) showed no differences in terms of (B) HA ataxin-3 aggregate numbers and (C) cerebellar Purkinje cell numbers compared to control and non-injected mice. (n = 6 per group; one-way ANOVA followed by post-hoc Bonferroni multiple comparison test.) Values ​​are expressed as mean ± SEM. [Figure 22] Injection of lentiviral particles encoding G3BP1 in SCA3 transgenic mice preserves the molecular layer of the cerebellum. SCA3 transgenic mouse models expressing a truncated form of ataxin-3 protein containing 69 glutamines were stereotactically injected into the cerebellum with lentiviral particles encoding GFP (control group) or with lentiviral particles encoding G3BP1 (treated group). (A) Representative cresyl violet stained images of brain sections of mouse cerebella treated with lentiviral particles encoding GFP (control) or with lentiviral particles encoding G3BP1. Upper panel: transduced lobe. Lower panel: non-transduced lobe. Scale: 200 μm. (B) G3BP1 treatment prevented cerebellar molecular layer shrinkage (lobe II / III) when compared to non-injected mice. (C) Non-transduced leaves (leaf IV / V) showed no differences in molecular layer thickness (n=6 per group; one-way ANOVA followed by post-hoc Bonferroni multiple comparison test). Values ​​are expressed as mean ± SEM. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] The present disclosure relates to isolated or artificial nucleotide sequences encoding GTPase-activating protein-binding protein 1 (G3BP1) for use in medicine, preferably in the treatment of polyglutamine diseases. Furthermore, the present invention also relates to such sequences, host cells comprising such vectors, vectors comprising the protein G3BP1, or compositions thereof, for use in medicine, preferably in the treatment of polyglutamine diseases.

[0032] In one embodiment, the isolated or artificial sequence encoding the protein GB3BP1 can be selected from the list shown in Table 1.

[0033] [Table 1-1] [Table 1-2]

[0034] In order to facilitate a better understanding of the present invention, certain terms are first defined. Furthermore, it should be noted that when a value or range of values ​​for a parameter is described, intermediate values ​​and ranges of the described values ​​are also intended to be part of the present invention. In this specification, the articles "a" and "an" are used to refer to one or more than one (i.e., at least one) of the grammatical object of the article. As an example, "element" means one element or more than one element, e.g., a plurality of elements. The term "comprises" is used herein to mean and is used in the same sense as the phrase "including, but not limited to." The term "or" is used herein to mean and is used in the same sense as the term "and / or," unless the context clearly indicates otherwise. For example, "sense strand or antisense strand" is understood to mean "sense strand or antisense strand, or sense strand and antisense strand." The term "about" is used herein to mean within a typical range of tolerance in the art. For example, "about" can be understood to mean about two standard deviations from the mean. In some embodiments, it means ±10% of the mean. In some embodiments, it means ±5% of the mean. When "about" is present before a series of numbers or a range, it is understood that "about" can modify each number in the series or range. The term "at least" before a number or series of numbers is understood to include the number adjacent to the term "at least" and all subsequent numbers or integers that can be logically included, as is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 15 nucleotides of a 21-nucleotide nucleic acid molecule" means that 15, 16, 17, 18, 19, 20, or 21 nucleotides have the indicated property. When at least is present before a series of numbers or a range, it is understood that "at least" can modify each number in the series (series) or range.

[0035] In the context of the present invention, the terms "treatment," "treat," or "treating" are used herein to characterize a therapeutic method or process that aims to (1) slow or halt the progression, worsening, or deterioration of the symptoms of the disease state or condition to which such term applies; (2) bring about a relief or amelioration of the symptoms of the disease state or condition to which such term applies; and / or (3) reverse or cure the disease state or condition to which such term applies.

[0036] As used herein, the term "subject" or "patient" refers to animals, preferably mammals, and more preferably humans (including adults and children). However, the term "subject" can also refer to non-human animals, particularly mammals such as mice, and non-human primates.

[0037] As used herein, the term "gene" refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular polypeptide or protein after being transcribed or translated.

[0038] As used herein, the term "coding sequence" or "sequence encoding a particular protein" means a nucleic acid sequence that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide, either in vitro or in vivo, when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences.

[0039] "G," "C," "A," "T," and "U" generally represent nucleotides that contain guanine, cytosine, adenine, thymidine, and uracil as the bases, respectively.

[0040] In one embodiment, the present invention describes an isolated or artificial sequence or a variant thereof for use in medicine.

[0041] Variants include naturally occurring variants due to, for example, allelic variation between individuals (e.g., polymorphisms), alternative splicing forms, etc. The term "variant" also includes G3BP1 gene sequences from other sources or organisms. Variants are preferably substantially homologous to one of SEQ ID NOs: 1-7, i.e. typically exhibit at least 90%, preferably at least 95%, more preferably at least 98%, more preferably at least 99% nucleotide sequence identity with one of SEQ ID NOs: 1-7.

[0042] Methods for aligning sequences for comparison are well known in the art, and such methods include GAP, BESTFIT, BLAST, FASTA, and TFASTA. GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find a global (sequence-wide) alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. The BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates the percentage of sequence identity and performs a statistical analysis of the similarity between two sequences. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI). Global percentages of similarity and identity can also be determined using one of the methods available in the MatGAT software package (Campanella et al., BMC Bioinformatics. 2003 Jul 10; 4:29. MatGAT: an application that generates similarity / identity matrices using protein or DNA sequences). As will be apparent to those skilled in the art, minor manual editing can be performed to optimize alignment between conserved motifs. The sequence identity values ​​presented in the present subject matter as percentages were determined over the entire amino acid sequence using BLAST with default parameters.

[0043] In one embodiment, the vector used according to the present invention is a non-viral vector.Typically, the non-viral vector can be a plasmid encoding GB3BP1.This plasmid can be administered directly or via liposome, exosome or nanoparticle.

[0044] Viral Vectors Gene delivery viral vectors useful in the practice of the present invention can be constructed using methodologies well known in the art of molecular biology. Typically, a viral vector carrying a transgene is assembled from a polynucleotide encoding the transgene, appropriate regulatory elements, and elements required for the production of viral proteins that mediate cell transduction.

[0045] The terms "gene transfer" or "gene delivery" refer to methods or systems for reliably inserting foreign DNA into a host cell. Such methods can result in the transient expression of non-integrated transferred DNA, the extrachromosomal replication and expression of transferred replicons (e.g., episomes), or the integration of the transferred genetic material into the genomic DNA of the host cell.

[0046] According to one embodiment, examples of viral vectors include adenovirus, lentivirus, retrovirus, herpes virus and adeno-associated virus (AAV) vectors.

[0047] Such recombinant viruses can be produced by transfecting packaging cells or by transient transfection with helper plasmids or viruses, using techniques known in the art.Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc.Detailed protocols for producing such replication-defective recombinant viruses can be found, for example, in WO95 / 14785, WO96 / 22378, US5,882,877, US6,013,516, US4,861,719, US5,278,056, and WO94 / 19478.

[0048] In a preferred embodiment, a lentiviral vector is used.

[0049] Lentiviral vectors are typically generated by transcomplementation in packaging cells co-transfected with a plasmid containing the vector genome and a packaging construct encoding only the proteins essential for lentiviral assembly and function. Self-inactivating (SIN) lentiviral vectors can be generated by eliminating the intrinsic promoter / enhancer activity of the HIV-1 LTR, which reduces the possibility of aberrant expression of cellular coding sequences located adjacent to the vector integration site (see, e.g., Vigna et al., J. Gene Med., 2: 308-316 (2000); Naldini et al., Science, 272: 263-267 (1996); and Matrai et al., Molecular Therapy, 18(3): 477-490 (2010)). The most common procedure for generating lentiviral vectors is to co-transfect a cell line (e.g., 293T human embryonic kidney cells) with the lentiviral vector plasmid and three packaging constructs encoding the viral Gag-Pol, Rev-Tat, and envelope (Env) proteins.

[0050] Vector Delivery The method of delivery or administration of viral vectors to neurons and / or astrocytes and / or oligodendrocytes and / or microglia generally includes any method suitable for delivery of vectors to selected synaptically connected cell populations, either directly or via hematopoietic cell delivery, so that at least a portion of the cells of said population are delivered.Vector can be delivered to any cell of the central nervous system, the peripheral nervous system, or both.Preferably, vector is delivered to brain cells.Generally, vector is delivered to brain cells, including, for example, brainstem (medulla, pons, and midbrain), cerebellum, substantia nigra cortex, striatum (caudate and putamen), frontotemporal lobes, visual cortex, spinal cord, or combinations thereof, or preferably any suitable subpopulations thereof.

[0051] Additional routes of administration can also include local application of the vector under direct visualization, for example, superficial cortical application, intranasal application, or other non-stereotactic application.

[0052] The target cells of the vectors of the present invention are cells of the brain of a subject affected by PolyQ SCA, preferably neuronal cells. Preferably, the subject is a human, generally an adult, but may be a child or infant.

[0053] The present invention also encompasses the delivery of vectors to biological models of the disease.In this case, the biological model can be any mammalian animal at any stage of development during parturition, for example, embryo, fetus, infant, juvenile or adult, preferably adult.Furthermore, the target cell can be from essentially any source, especially non-human primates and mammals of rodent (mouse, rat, rabbit, hamster), carnivora (cat, dog) and other non-human systems (e.g. zebrafish model system) of artiodactyla (cow, pig, sheep, goat, horse).

[0054] Preferably, the method of the present invention includes intracerebral administration by stereotactic injection. However, other known delivery methods can also be adapted according to the present invention. For example, for broader distribution of the vector across the brain, it can be injected into the cerebrospinal fluid, for example, by lumbar puncture, cisterna magna (posterior cerebellum-medullary cistern) or ventricular puncture. To direct the vector to the brain, it is injected into the spinal cord or peripheral ganglia, or into the flesh (subcutaneous or intramuscular) of the body part of interest. In some situations, the vector can be administered via an intravascular approach. For example, the vector can be administered intra-arterially (carotid artery) in situations where the blood-brain barrier is impaired. Furthermore, for more global delivery, the vector can be administered during the "opening" of the blood-brain barrier, which is achieved by injection of hypertonic solutions, including mannitol or ultrasound local delivery.

[0055] The vectors used herein can be formulated in any suitable vehicle for delivery. For example, they can be placed in pharmaceutically acceptable suspensions, solutions or emulsions. Suitable vehicles include saline and liposomal formulations. More specifically, pharmaceutically acceptable carriers can include sterile aqueous solutions of non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline, buffered media. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (e.g., based on Ringer's dextrose), and the like.

[0056] Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

[0057] Colloidal dispersion systems can also be used for targeted gene delivery, including macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes or exosomes.

[0058] The present invention is further illustrated by the following examples, which, however, should not be construed as limiting the scope of the present invention.

[0059] Working Example Materials and Methods Plasmid vectors In one embodiment, plasmids encoding human ataxin-3 contain 28 glutamines (pEGFP-C1-Ataxin3Q28; #22122; Addgene) or 84 glutamines (pEGFP-C1-Ataxin3Q84; #22123; Addgene), gifts from Henry Paulson, both fused to the GFP protein at the N-terminus. 18 Plasmids encoding human ataxin-2 containing 22 glutamines (pEGFP-Ataxin2Q22) or 104 glutamines (pEGFP-Ataxin2Q104) were kindly provided by Professor Stefan Pulst. 19 The LacZ gene was cloned in our laboratory under the control of the phosphoglycerate kinase promoter (PGK). 20 , GFP constructs were cloned as previously described 21 A plasmid encoding human G3BP1 (SEQ ID NO:1) purchased from Source Bioscience was cloned into a lentiviral vector backbone using Gateway™ LR Clonase™ II Enzyme Mix, Invitrogen, following the manufacturer's instructions. G3BP1-ΔNTF2 (G3BP1 deleted at sites 11-133) and G3BP1-ΔRRM (G3BP1 deleted at sites 340-415) constructs were synthesized from GeneScript and cloned into vector pcDNA3.1+N-MYC. Validated shRNA targeting mouse G3BP1 (#MSH031039-LVRU6MP-b) and shRNA scramble were obtained from GeneCopoeia (USA) as controls (no known target, #CSHCTR001-LVRU6MP).

[0060] Lentiviral vector containing a plasmid encoding human G3BP1 In one embodiment, a plasmid encoding human G3BP1 (one of SEQ ID NOs: 1-7) was cloned into a self-inactivating lentiviral vector under the control of the PGK promoter using Gateway™ LR Clonase™ II Enzyme Mix, Invitrogen, according to the manufacturer's instructions. The lentiviral vector was produced in HEK (human embryonic kidney) 293T cells using the four-plasmid system previously described. 25 Viral production was quantified using a RetroTek HIV-1 p24 antigen enzyme-linked immunosorbent assay (ELISA) (ZeptoMetrix) according to the manufacturer's instructions.

[0061] GB3BP1 mutagenesis of serine 149 residue In one embodiment, site-directed mutagenesis was performed using the NZY mutagenesis kit (NZYTech) according to the manufacturer's instructions. In the human mutant of G3BP1 (GeneBank accession: DQ893058.2), the serine was changed to alanine or aspartic acid at site 149 to generate a G3BP1 phosphorylation-dead mutant (G3BP1_S149A) or a G3BP1 phosphorylation-mimicking mutant (G3BP1_S149D), respectively. The pair of primers used to induce the substitution S149A was as follows: SEQ ID NO:8: 5'-CT GAG CCT CAG GAG GAG GCT GAA GAA GAA GTA GAG-3' and SEQ ID NO:9: 5'-CT CTA CTT CTT CTT CAG CCT CCT CCT GAG GCT CAG-3'. The pair of primers used to derive substitution S149D was as follows: SEQ ID NO:10: 5'-CT GAG CCT CAG GAG GAG GAT GAA GAA GAA GTA GAG-3' and SEQ ID NO:11: 5'-CTC TAC TTC TTC TTC ATC CTC CTC CTG AGG CTC AG-3'. Mutations S149A and S149D were confirmed by DNA sequencing (Eurofins Genomics).

[0062] Neuroblastoma culture and transfection In one embodiment, mouse neuroblastoma cell line (Neuro2a cells) obtained from the American Type Culture Collection cell biology bank (CCL-131) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v / v) fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin. Cells were seeded in 12- or 6-multiwell plates. After 24 h of growth, cells were transfected at a DNA concentration of 0.5 μg per well using polyethylenimine reagent (PEI; PEI MAX Polysciences, Inc.) according to the manufacturer's instructions. For SG induction experiments, cells were treated with sodium arsenite (SA, Sigma Aldrich 10 μg / mL) at a final concentration of 0.05 M for 1 h before harvesting.

[0063] Human fibroblast cell culture In one embodiment, patient fibroblasts from SCA2, SCA3, and healthy individuals were obtained from the Coriell Institute or kindly provided by collaborators. 22 , fully characterized for CAG expansion: SCA2 (patient 1: 22 / 41; patient 2: 20 / 44); SCA3 (patient 1: 18 / 79; patient 2: 22 / 77; patient 3: 23 / 80; patient 4: 23 / 71; patient 5: 24 / 74); healthy controls (1: 14 / 19; 2: 14 / 23; 3: 22 / 23; 4: 22 / 23). Fibroblasts were maintained in culture in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% (v / v) fetal bovine serum (FBS), 100 U / mL penicillin and 100 μg / mL streptomycin. All cell cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO2.

[0064] Translation rate assay (SUnSET protocol) In one embodiment, a method was used that allows monitoring and quantification of global protein synthesis based on the incorporation of puromycin during translation. N2a cells were plated in multi-well plates and transfected with lacZ or G3BP1. 24 hours after transfection, cells were incubated with 10 mg / ml puromycin (Sigma) for 15 minutes and then harvested for Western blot processing. As a positive control for translation inhibition, some cells were incubated with 10 mM cycloheximide (CHX, Sigma) for 15 minutes and then incubated with 10 mg / ml puromycin (Sigma) for another 15 minutes. For stress granule conditions, cells were treated with 0.05 M sodium arsenite for 1 hour and then incubated with 10 mg / ml puromycin (Sigma) for another 15 minutes. In addition, a control of untreated cells was also used.

[0065] Human Brain Tissue In one embodiment, postmortem striatal and cerebellar brain tissues from clinically and genetically confirmed SCA2 patients were obtained from the NIH NeuroBioBank (USA). Control striatal and cerebellar tissues from healthy individuals with no diagnosed neurological condition were obtained from the NIH NeuroBioBank (USA). Tissues stored in 4% PFA solution were dehydrated in 30% sucrose / PBS for 48 hours, cryoprotected at -80°C, dissected into 40 μm slices using a cryostat (Cryostar NX50, ThermoFisher Scientific), and stored in free-floating PBS / sodium azide solution at 4°C.

[0066] animal In one embodiment, C57BL / 6J wild type adult and transgenic SCA3 mice bred in the animal facility of the Universidade do Algarve. 23The animals were maintained in a temperature-controlled room with a 12-h light-12-h dark (fark) cycle. Food and water were distributed ad libitum. All experiments were performed in accordance with the European Communities Council Directive (86 / 609 / EEC) on the care and use of laboratory animals. The researchers received accredited training (FELASA course) from the Portuguese authorities (Direccao Geral de Alimentacao e Veterinaria) in the project Neuropath (421 / 2019) and approval to carry out the experiments.

[0067] Lentiviral Vectors In one embodiment, cDNAs encoding human G3BP1, GFP, ATXN2MUT, and ATXN3MUT were cloned into a self-inactivating lentiviral vector under the control of the PGK promoter, as previously described. 24 Lentiviral vectors were produced in HEK (human embryonic kidney) 293T cells using the four-plasmid system previously described. 25 Viral production was quantified using a RetroTek HIV-1 p24 antigen enzyme-linked immunosorbent assay (ELISA) (ZeptoMetrix) according to the manufacturer's instructions.

[0068] In vivo injection of lentiviral vectors In one embodiment, for stereotactic injection of lentiviral vectors, concentrated viral stocks were thawed on ice and homogenized. Animals were anesthetized by intraperitoneal injection (IP) of a mixture of ketamine (75 mg / kg, Nimatek, Dechra) and medetomidine (0.75 mg / kg, DOMTOR®, Esteve). For the SCA2 lentiviral mouse model, mice (10-12 weeks old) were injected with lentiviral particles encoding human ATXN2MUT containing 82 glutamines or lentiviral particles encoding ATXN2MUT and G3BP1 into the left and right hemispheres of the striatum, respectively, according to the following brain coordinates relative to bregma: Anterior-Posterior (+0.6), Medial-Lateral (+ / -1.8), Dorsal-Ventral (-3.3). 26 A lentiviral concentration of 400ng p24 / μl was injected at a rate of 0.20μl / min. For SCA3 lentiviral mice, viral particles encoding human ATXN3MUT or ATXN3MUT and G3BP1 containing 72 glutamines were injected into the mouse striatum (left and right hemispheres, respectively) at 400ng p24 / ml, using the same coordinates as above. To perform safety assays, wild-type C57 / BL6 mice (10-12 weeks old) were injected into the striatum with lentiviral particles encoding G3BP1 at a lentiviral concentration of 400ng p24 / ul, using the same coordinates as above. For transgenic animals, lentiviral particles encoding G3BP1 or GFP, as respective controls, were injected into mouse cerebellum (4 weeks old) with 800 ng p24 / μl lentivirus at the following coordinates: −1.6 mm rostral to lambda, 0.0 mm midline, and −1.0 mm ventral to the skull surface, with mouse bar set at −3.3. 21For G3BP1 silencing studies in SCA2, wild-type C57 / BL6 mice (10–12 weeks old) were injected in the striatum with lentiviral particles encoding human ATXN2MUT containing 82 glutamines and lentiviral particles encoding shRNA scramble, whereas in the contralateral hemisphere, lentiviral particles encoding human ATXN2MUT containing 82 glutamines and lentiviral particles encoding shRNA targeting mouse G3BP1 were injected. For SCA3, the procedure was similar but with lentiviral particles encoding human ATXN3MUT containing 72 glutamines. Lentiviral particles were injected at a lentiviral concentration of 400 ng p24 / ul using the same coordinates as above. All stereotactic injections were performed by an autoinjector (Stoelting Co.) with a 34-gauge blunt-end needle coupled to a Hamilton syringe. Mice were sacrificed a few weeks after surgery for posterior analysis: according to the model, SCA2 lentiviral mice: 4 weeks and 12 weeks; SCA3 lentiviral mice: 4 weeks; G3BP1-injected mice: 4 weeks; SCA3 transgenic mice: 9 weeks.

[0069] Behavioral testing In one embodiment, transgenic mice were subjected to several motor behavioral tests before stereotaxic injection (at 4 weeks of age) starting every 3 weeks until 9 weeks after injection. Locomotor and gait coordination (gait) were blindly assessed by rotarod and footprint tests, following the same methods as previously described. 21 In the analysis of the footprint test, steps taken by the mice at the beginning and end of the walking test are not included and are not considered for the measurements. Swimming performance was assessed by placing the mice at one end of a rectangular tank (100 × 10.5 × 20 cm) filled with room temperature water. The mice were allowed to swim freely for 1 m until they reached the platform and the time taken to cross the tank was recorded. The mice performed three trials per trial, with an interval of 15–20 min between each trial. The average value of the time taken to cross the tank in the tree trial was used for statistical analysis.

[0070] Tissue processing In one embodiment, animals were sacrificed by sodium pentobarbital overdose and perfused transcardially with 0.1 M phosphate buffer and 4% paraformaldehyde fixative (Sigma Aldrich) for immunohistochemical assays, or had cervical dislocation and brain striatal punches for qPCR and Western blot analysis using a 2.5 mm diameter Harris Core pen (Ted Pella Inc.). Brains and striatal punches were fixed in 4% paraformaldehyde for 24 h, then dehydrated in 30% sucrose / 0.1 M phosphate buffer (PBS) for 48 h and cryoprotected at −80° C. Sagittal or coronal brain sections of 30 μm and 25 μm, respectively, were obtained using a cryostat-microtome model CryoStar NX50 (Thermofisher). For storage, brain slices were stored suspended in 0.02% (w / v) sodium azide in PBS at 4°C.

[0071] Cresyl violet staining In one embodiment, to stain the brain slices with cresyl violet, they were mounted on gelatin-coated microscope slides. The brain slices were successively immersed in water, 96% (v / v) ethanol, 100% (v / v) ethanol, xylene, 75% (v / v) ethanol, and 0.1% (w / v) cresyl violet solution. To wash the slices, the brain slices were successively immersed in water, 75% (v / v) ethanol, 96% (v / v) ethanol, 100% (v / v) ethanol, and xylene. Finally, the brain slices were mounted with Eukitt (Sigma-Aldrich). Images were acquired with a 10x objective on a Zeiss Axio Imager Z2.

[0072] immunocytochemistry In one embodiment, for immunocytochemical procedures, cells were fixed using 4% paraformaldehyde (PFA) fixative solution for 20 min and washed with 0.1 M phosphate buffered saline (PBS). Samples were then incubated in PBS containing 0.1% Triton™ X-100 for 10 min. Blocking with PSB containing 1% bovine serum albumin (Sigma) was performed for 30 min. Samples were incubated with primary antibodies at appropriate dilutions at 4oC overnight and with secondary antibodies (1:200) at room temperature for 2 h. Secondary antibodies were conjugated to fluorophores (Alexa Fluor®, Invitrogen). Finally, coverslips were mounted on microscope slides using Fluoromount-G mounting medium with DAPI (Invitrogen).

[0073] immunohistochemistry In one embodiment, the immunohistochemistry procedure for optical imaging began with incubation of brain sections in phenylhydrazine diluted in phosphate buffer (1:1000; 15 min, 37° C.). For human brain sections, an additional step was performed with Tris-buffered saline pH 9 antigen retrieval method (30 min, 95° C.). Brain sections were blocked in 10% normal goat serum in 0.1% Triton™X phosphate buffer (1 h, room temperature), incubated with primary (overnight at 4° C.) and secondary biotinylated antibodies (2 h at room temperature), respectively, diluted in blocking solution, and then reacted with Vectastain elite avidin-biotin-peroxidase kit and 3,3′-diaminobenzidine substrate (both from Vector Laboratories). Sections were then collected on microscope slides, dehydrated in increasing concentrations of ethanol solutions (75, 96, and 100%) and xylene, and finally coverslipped with the mounting medium Eukitt (O. Kindler GmbH & CO). For fluorescent immunohistochemistry, brain sections were incubated in the blocking solution described above, followed by primary and secondary antibody incubation. Brain sections were mounted on microscope slides with Fluoromount-G Mounting medium supplemented with 4',6-diamidino-2-phenylindole (DAPI) (Invitrogen).

[0074] immunochemical antibodies In one embodiment, for the immunochemical procedures the following primary antibodies were used: Mouse anti-ataxin-2 (1:1000, ref. 611378, BD Biosciences), mouse anti-ubiquitin (1:1000, ref. 3936S, Cell Signaling), rabbit anti-DARPP-32 (1:1000, ref. AB10518, Merck Millipore), rabbit anti-G3BP1 (1:1000, ref. 07-1801, Millipore), mouse anti-human G3BP1 (1:1000, ref. 611126, BD Biosciences), anti-G3BP1 (1:1000, ref. 05-1938; Sigma-Aldrich), mouse anti-GFAP (1:1000, ref. 644702, BioLegend), rabbit anti-HA (1:1000, ref. Ab9110, Abcam), mouse anti-calbindin D‐28K (1:1000, ref. C9848, Sigma Aldrich), mouse anti-PABP‐1 (1:1000, ref. 04‐1467, Millipore), and mouse β‐Gal (14B7) (1:500, ref. 2372, Cell Signaling Technology).

[0075] Image Quantitative Analysis and Data Processing In one embodiment, immunocytochemistry images were acquired on a Zeiss Axio Imager Z2 for quantification and a Zeiss LSM710 confocal microscope for representative images. Quantitative analysis was performed blinded by counting the number of cells with aggregates in 100 transfected cells using a 40x or 63x objective in each independent experiment. Immunohistochemistry images from lentiviral mouse models were acquired with a 20x objective on a Zeiss Axio Imager Z2 and Axio Scan Z1 slide scanner microscope. To quantify ataxin-2 aggregates and DARP-32 staining loss, 18 coronal sections per animal were analyzed with ZEN light software (Zeiss) and a complete rostrocaudal picture of the striatum was obtained. Ataxin-2 inclusions were manually counted in all animals. DARP-32 neuronal lesion areas were measured manually for all animals, allowing quantification of the depletion volume according to the following formula: volume = d * (a1 + a2 + a3), where d is the distance between consecutive sections (200 μm) and a1 + a2 + a3 are the depletion areas of individual sections. Immunohistochemistry images from transgenic mouse animals were acquired by acquiring eight sagittal sections spanning 280 μm throughout the cerebellum, stained with anti-HA, anti-calbindin, and DAPI on a Zeiss Axio Imager Z2 microscope using a 20x objective. For each section, the number of cells with HA aggregates and Purkinje cells in all cerebellar lobules was counted in a blinded fashion using image analysis software (ZEN 2.1 lite, Zeiss).

[0076] Western blotting In one embodiment, samples were lysed in 10x RIPA solution (Merck Millipore) for cell extracts or homogenized in urea / DTT solution for mouse striatal punches, both containing a cocktail of protease inhibitors (Roche), followed by 5 cycles of ultrasonic sonication (Bioruptor Pico) for 30 seconds on, 30 seconds off. Protein concentration levels were measured using the Pierce™ BCA Protein Assay Kit (Thermo Scientific) for cell lysates and NZYBradford reagent (Nzytech) for mouse samples. Protein extracts were separated on SDS-polyacrylamide gels (7.5% and 12%), then proteins were transferred to PVDF membranes (Merck Millipore), membranes were blocked with 3% BSA or 5% milk in TBS-T, and antibody probed for primary overnight at 4°C and secondary overnight at room temperature for 2 hours. The following antibodies were used: mouse anti-ataxin-2 (1:1000, ref. 611378, BD Biosciences); mouse anti-ataxin-3 (1H9) (1:1000, ref. MAB5360, BD Biosciences); rabbit anti-G3BP1 (1:1000, ref. 07-1801, Millipore); mouse anti-human G3BP1 (1:1000, ref. 611126, BD Biosciences); anti-G3BP1 (1:1000, ref. 05-1938; Sigma-Aldrich); mouse anti-β-actin (1:5000, ref. A5316, Sigma Aldrich) mouse anti-B-tubulin (1:5000, ref. T7816, Sigma); mouse anti-puromycin (1:250, ref. MABE343, Millipore); mouse anti-GFP (1:1000, ref. 668205, BioLegend); mouse β-Gal (14B7) (1:500, ref. 2372, Cell Signaling Technology).The membranes were resolved using Enhanced Chemiluminescence (GE Healthcare) and scanned with a ChemiDoc™ XRS+ (Bio-Rad). Optical densitometry analysis was performed using Image J software.

[0077] RT‐qPCR According to one embodiment, total RNA from mouse striatal punches was initiated by Trizol (Invitrogen) tissue dissociation and RNA / DNA / protein chloroform separation. Both mouse and cell samples were then extracted using the NZY Total RNA Isolation kit (Nzytech). RNA concentration and purity were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). cDNA molecules were produced for 1 μg of RNA using the iScript cDNA Synthesis Kit (Bio-Rad) according to the manufacturer's recommendations. Quantitative RT-qPCR was performed on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) using homemade primers for the genes of interest and the human GAPDH housekeeping gene as a control, with SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad). mRNA expression levels relative to mRNA gene controls were measured using amplification values. The following primers were used: human ATXN2 (QT01852480) and human ATXN3 (QT00094927) QuantiTect Primer Assays, Qiagen human G3BP1 (forward SEQ ID NO:12: 5'-GAA ATC CAA GAG GAA AAG CC-3'; reverse SEQ ID NO:13: 5'-CCC AAG AAA ATG TCC TCA AG), human GAPDH (forward SEQ ID NO:14: 5'-ACA GTT GCC ATG TAG ACC-3'; reverse SEQ ID NO:15: 5'-TTG AGC ACA GGG TAC TTT A-3') and mouse Hprt (forward SEQ ID NO:16: 5'-AGG GAT TTG AAT CAC GTT TG-3; reverse SEQ ID NO:17: 5'-TTT ACT GGC AAC ATC AAC AG-3') KiCqStart Pre-designed Primers, Sigma-Aldrich.

[0078] statistical analysis In one embodiment, statistical analysis was performed using either the Student t test or one-way ANOVA complemented with the Bonferroni multiple comparison test, relying on GraphPad software (La Jolla).

[0079] result Stress granule assembly does not alter ATXN2 and ATXN3 protein levels. SGs are cellular foci that form in response to stress, where mRNAs, translation factors, and RBPs coalesce to prevent cellular injury. 27,28 Therefore, we investigated the impact of SG assembly on ATXN2 and ATXN3 protein dynamics in both pathological (ATXN2MUT and ATXN3MUT) and non-pathological forms (ATXN2WT and ATXN3WT). To this end, we pharmacologically induced SG assembly using sodium arsenite in Neuro2a cells expressing ATXN2 (ATXN2WT: pEGFP-ATXN2-Q22 or ATXN2MUT: pEGFP-ATXN2-Q104) or ATXN3 (ATXN3WT: pEGFP-ATXN3-Q24 or ATXN3MUT: pEGFP-ATXN3-Q844) (Figure ​(Figure9a). 9a). As previously reported, ataxin-2 is recruited to SGs, but 29 In contrast, ataxin-3 is not recruited. Expression of mutants of both proteins leads to the formation of aggregates (Fig. 1a, 1f). However, SG assembly did not alter the number of cells with ATXN2MUT or ATXN3MUT aggregates compared to control conditions in which no stress stimuli were induced (ATXN2MUT and ATXN3MUT, respectively) (Fig. 1c, 1h). Although non-pathological forms of the proteins do not form aggregates, when SG assembly is induced, aggregate-like structures are formed in both ATXN2WT and ATXN3WT conditions (Fig. 1a, 1f). SG assembly is accompanied by phosphorylation of eiF2α and translational repression. 30, reducing total protein synthesis (Fig. 9b, c). We therefore investigated whether ATXN2 and ATXN3 protein levels were altered by SG assembly and analyzed their levels by Western blotting (Fig. 1b, 1g). In agreement with the results observed for the aggregates, no changes in the levels of these proteins were observed between the different experimental conditions, nor in the non-pathological (Fig. 1d, 1i) and pathological (Fig. 1e, 1j) protein forms. Taken together, these results indicated that SG assembly reduces global translation but does not seem to interfere with ATXN2 and ATXN3 aggregation, nor their protein levels.

[0080] Overexpression of G3BP1 reduces the number of cells with aggregates and the levels of ATXN2 and ATXN3 proteins SG assembly can also be induced by overexpression of its core components, including G3BP1. 13,31 , an RBP capable of both mRNA stabilization and degradation 15However, in this disclosure, we observed that Neuro2a G3BP1 overexpression alone was less effective in inducing SG formation than when combined with sodium arsenite stimulation (Figure 10). In this line, fibroblasts from SCA2 and SCA3 patients showed diffuse expression of G3BP1, which is also observed in healthy fibroblasts (Figure 11). Conversely, upon sodium arsenite treatment, G3BP1 condenses within the positive foci (Figure 11). As observed in SGs, overexpression of G3BP1 also leads to inhibition of protein synthesis, albeit at a lower level (Figures 9b, 1d). Taking this into account, we next investigated the impact of G3BP1 overexpression on ATXN2MUT and ATXN3MUT proteins. To achieve this goal, we co-transfected Neuro2a cells with ATXN2MUT or ATXN3MUT and G3BP1, and used control cells co-transfected with ATXN2MUT or ATXN3MUT and lacZ, and cells transfected with ATXN2MUT or ATXN3MUT (Fig. S12). As mentioned above, expression of mutants of both proteins in Neuro2a leads to the formation of aggregates, which are characteristic of polyQ disease (Fig. S2a, b). We found that G3BP1 overexpression could significantly reduce the number of cells with both ATXN2MUT (ATXN2MUT+G3BP1: 0.39 ± 0.0153, vs. ATXN2MUT: 0.53 ± 0.036, n = 3, P = 0.0233) and ATXN3MUT (ATXN3MUT+G3BP1: 0.35 ± 0.036, vs. ATXN3MUT: 0.66 ± 0.073, n = 3, P = 0.0201) aggregates compared to control conditions (Figures 2c, 2d). We next examined whether the reduction in aggregates observed upon G3BP1 overexpression was associated with reduced protein levels of ATXN2MUT and ATXN3MUT (Figures 2e, 2f). Additionally, the effect of G3BP1 overexpression on protein levels in the non-pathological forms ATXN2WT and ATXN3WT (Figures 2e, 2f), respectively, was also analyzed.We found that G3BP1 overexpression could significantly reduce the expression levels of both ATXN2WT (ATXN2WT+G3BP1: 0.65 ± 0.06, vs. ATXN2WT+lacZ: 0.692 ± 0.08, n = 5, P = 0.04) and ATXN2MUT (ATXN2MUT+G3BP1: 0.35 ± 0.1343, vs. ATXN2MUT+lacZ: 0.82 ± 0.116, n = 5, P = 0.0076) (Fig. 2g, 2h). Similarly, a significant decrease in ATXN3WT and ATXN3MUT levels was also observed upon G3BP1 overexpression compared to control conditions (ATXN3WT+G3BP1: 0.608 ± 0.026, vs. ATXN3WT+lacZ: 0.3 ± 0.071, n = 5, P = 0.02 and ATXN3MUT+G3BP1: 0.28 ± 0.067, vs. ATXN3MUT+lacZ: 0.95 ± 0.154, n = 5, P = 0.004) (Fig. 2i, 2j, respectively). However, no changes were observed in mouse endogenous levels of Ataxin-2 and Ataxin-3 upon G3BP1 overexpression (Fig. S13). Furthermore, in additional control experiments, GFP levels were not altered when G3BP1 was overexpressed (Fig. S14). Overall, these results indicate that G3BP1 reduces the levels and aggregation of mutant ataxin-2 and mutant ataxin-3 proteins. The NTF2-like domain is important for G3BP1 action on ataxin-2 and ataxin-3 mutant proteins.

[0081] GB3BP1 binds to mRNA and DNA 32 , an RBP with several molecular and biological functions, including helicase, and has important functions in immune responses 34 In general, RBPs, including GB3BP1, interact with mRNAs through specific RNA-binding domains. 35,36 The RNA recognition motif (RRM) of G3BP1 is known to interact with target RNA sequences. 37 G3BP1 also contains an NTF2-like domain that is involved in shuttling proteins into the nucleus through the nuclear pore complex. 38 , promoting protein-protein interactions 39, which mediates G3BP1 dimerization and is important for SG biogenesis 13Therefore, to better understand the effect of G3BP1 on the aggregation and abundance of mutant ataxin-2 and ataxin-3, we developed two different forms of the protein, one with a deletion of the NTF2-like domain (G3BP1-ΔNTF2) and the other with a deletion of the RRM domain (G3BP1-ΔRRM) (Fig. 3a, 3b). We then co-transfected Neuro2a cells with either ATXN2MUT or ATXN3MUT and G3BP1-ΔNTF2 or G3BP1-ΔRRM, as well as with full-length G3BP1 and lacZ as controls (Fig. 3c, 3d). Expression of G3BP1‐ΔRRM results in a significant decrease in the number of cells with ATXN2MUT and ATXN3MUT+lacZ aggregates compared to the lacZ control condition (ATXN2MUT+G3BP1‐ΔRRM: 55 ± 0.815 vs. ATXN2MUT+lacZ: 62 ± 0.814, n = 4, P < 0.001, and ATXN3MUT+G3BP1‐ΔRRM: 66.5 ± 2.305 vs. ATXN3MUT+lacZ: 80.7 ± 2.37, n = 4, P < 0.001) (Fig. ​(Fig.3e, ​(Fig.3f,3c). 3e, ​(Fig.3f). 3e, ​(Fig.3f). 3f). However, compared to expression of full-length G3BP1, G3BP1‐ΔRRM results in a significant increase in the number of cells with ATXN2MUT and ATXN3MUT aggregates. Conversely, expression of G3BP1-ΔNTF2 resulted in an increase in cells with aggregates of ATXN2MUT and ATXN3MUT compared to lacZ and full-length G3BP1 conditions (Fig. 3e, 3f).We next analyzed the levels of ATXN2MUT and ATXN3MUT upon expression of both truncated forms of G3BP1 (Fig. 3g, 3i). We found that G3BP1-ΔRRM significantly reduced the levels of ATXN2MUT and ATXN3MUT compared to the control (ATXN2MUT+G3BP1-ΔRRM: 0.48 ± 0.035 vs. ATXN2MUT+lacZ: 0.64 ± 0.013, n = 4, P < 0.001, and ATXN3MUT+G3BP1-ΔRRM: 0.725 ± 0.001 vs. ATXN3MUT+lacZ: 0.93 ± 0.012, n = 4, P < 0.001, respectively) (Fig. 3h, 3j). Conversely, expression of G3BP1-ΔNTF2 significantly increased the levels of ATXN2MUT and ATXN3MUT proteins (Fig. 3h, 3j).Taken together, these results point to a relevant role for the NTF2-like domain in the molecular mechanism of G3BP1 action on mutant ataxin-2 and mutant ataxin-3 proteins.

[0082] The Ser149 phosphorylation site is important for G3BP1 action on ataxin-2 and ataxin-3 mutant proteins In the G3BP1 protein, the NTF2-like domain is located close to the phosphorylation site (Ser-149) and may play an important functional role. 17,36G3BP1‐ΔRRM could reduce the levels and aggregation of ATXN2MUT and ATXN3MUT, but less than full-length G3BP1. Therefore, the inventors of this disclosure aimed to investigate the importance of Ser149 in the functional role of G3BP1. To that end, two phosphorylation-mimetic G3BP1s, phospho-mimetic S149D and non-phosphorylatable S149A, were developed (Figure 15). Neuro2a cells were co-transfected with ATXN2MUT or ATXN3MUT and G3BP1(S149D) and G3BP1(S140A). By confocal imaging, it was observed that in cells expressing wild-type G3BP1, no aggregates of ATXN2MUT or ATXN3MUT were present (Figure 4a, 4b; white arrows). The same pattern is observed with expression on phospho-mimetic G3BP1(S149D). On the other hand, in cells expressing unphosphorylated (phospho-dead) G3BP1(S149A), aggregates of ATXN2MUT and ATXN3MUT were observed (Figures 4a, 4b; white arrowheads). Next, we examined the effects of two types of phosphorylation compounds on the protein levels of ATXN2MUT and ATXN3MUT (Figures 4c, 4d). We found that the level of ATXN2MUT protein was significantly increased by G3BP1(S149A) expression (ATXN2MUT+G3BP1: 0.24±0.026 vs. ATXN2MUT+G3BP1(S149A): 0.37±0.028, n=3, P<0.05) (Figure 4e). On the other hand, the level of ATXN2MUT protein was similar between wild-type G3BP1 and phosphorylation-mimetic G3BP1(S149D) (Figure 4e). In the same lineage, the levels of ATXN3MUT protein are increased upon expression of non-phosphorylatable G3BP1(S149A) compared to wild-type G3BP1 and G3BP1(S149D) conditions (Figure 4g). Expression of G3BP1(S149D) also results in a significant reduction in the levels of ATXN3MUT compared to the wild-type G3BP1 condition (ATXN3MUT+G3BP1: 0.36±0.03 vs. ATXN3MUT+G3BP1(S149D): 0.25±0.01, n=3, P<0.05). A significant reduction in the mRNA levels of ATXN2MUT and ATXN3MUT was observed upon wild-type G3BP1 expression compared to the control condition (Figure 16).However, no difference was observed in the mRNA levels of ATXN2MUT and ATXN3MUT following expression of the two phosphorylation compounds compared with wild-type G3BP1 (Figures 4f,h). Overall, these results suggest that the Ser-149 phosphorylation site is important for G3BP1 molecular activity and regulates the aggregation and protein levels of mutant ataxin-2 and mutant ataxin-3.

[0083] G3BP1 mRNA and protein levels are reduced in SCA2 and SCA3, but its silencing increases aggregation in mouse brain Previous studies have reported that mutant PolyQ proteins can dysregulate the expression of several genes. 1,41 Indeed, the inventors herein have shown that expression of mutant ataxin-3 results in an abnormal decrease in the concentration of wild-type ataxin-2. 42We then analyzed the levels of G3BP1 in samples from SCA2 and SCA3 patients and disease models in this line. In autopsy brain specimens from SCA2 patients, we detected reduced immunodetection of G3BP1 in both the striatum and cerebellum compared to healthy controls (Figure 17). Furthermore, we detected significantly reduced levels of G3BP1 protein (Figures 5a, 5c) and mRNA (Figure 5d) in fibroblasts from SCA2 patients compared to fibroblasts from healthy controls. In the same line, we observed reduced G3BP1 protein (Figures 5b, 5e) and mRNA levels (Figure 5f) in fibroblasts from SCA3 patients compared to fibroblasts from healthy controls. This reduction was also observed in the transgenic mouse model of SCA3 used in this study (Figures 5g-i). In fact, G3BP1 protein and mRNA levels are significantly reduced in transgenic SCA3 animals compared to wild-type C57BL / 6. The transgenic mice express a truncated form of ataxin-3 with 69 glutamines in the Purkinje cells of the cerebellum. Indeed, the reduction of G3BP1 detected in the transgenic animals is particularly evident in these cells (Figure 18). To investigate the functional consequences of G3BP1 reduction, lentiviral vectors encoding a validated shRNA targeting G3BP1 (shG3BP1) (Figure 19) were injected into lentiviral rat models of SCA2 and SCA3. 43,44(Fig. 5i, 5l). Briefly, one side of the striatum was co-injected with lentiviral vectors encoding ATXN2MUT (or ATXN3MUT) and shG3BP1, and the contralateral hemisphere was injected with ATXN2MUT (or ATXN3MUT) and scrambled shRNA (shSrc) as a control. Four weeks after injection, animals were sacrificed and the striatum was histologically analyzed for the presence or absence of ATXN2MUT and ATXN3MUT aggregates (Fig. 5j, 5m). We found that silencing of G3BP1 led to a significant increase in the mean aggregate numbers in ATXN2MUT (ATXN2MUT+shScr: 434±55.62 vs. ATXN2MUT+shG3BP1: 228±98.85, n=4, P<0.01) and ATXN3MUT (ATXN3MUT+shScr: 390±26.89 vs. ATXN3MUT+shG3BP1: 290±22.37, n=3, P<0.05) (Figure 5k, 5n). Taken together, these results highlight that reduced levels of G3BP1 mRNA and protein are important for disease pathogenesis in SCA2 and SCA3.

[0084] Restoring G3BP1 levels ameliorates neuropathology in SCA2 and SCA3 lenticular mouse models Lentiviral vector-mediated expression of ATXN2MUT and ATXN3MUT is a neuropathological sign also seen in postmortem human tissues 45‐47 , formation of intraneuronal aggregates and loss of neuronal markers 43,44Thus, we investigated whether restoring G3BP1 levels would improve the neuropathological abnormalities induced by ATXN2MUT and ATXN3MUT in vivo. A lentiviral vector encoding ATXN2MUT (or ATXN3MUT) and human G3BP1 was coexpressed in one hemisphere of the striatum, and a lentiviral vector encoding ATXN2MUT (or ATXN3MUT) was injected into the contralateral hemisphere as a control (Fig. 6a, 6b). The animals were sacrificed 12 weeks after injection for the SCA2 lentiviral mouse model and 4 weeks after injection for the SCA3 lentiviral mouse model, and the striatum was histologically analyzed. In both models, expression of G3BP1 was able to significantly reduce the number of ATXN2MUT aggregates (ATXN2MUT+G3BP1: 1466 ± 31.13, n = 5, vs. ATXN2MUT: ± 71.04, n = 5, P = 0.0002) and ATXN3MUT aggregates (ATXN3MUT+G3BP1: 6066 ± 1958, vs. ATXN3MUT: 30076 ± 2717, n = 7, P < 0.0001) (Fig. 6c-d, 6f-g). ATXN2 and ATXN3 mRNA and soluble protein levels were also analyzed in the animal groups at 4 weeks after injection (Fig. 20). In the SCA2 lentiviral model, no significant differences were observed in the mRNA and protein levels of ATXN2MUT upon G3BP1 expression (Fig. 20a-b). In contrast, in the SCA3 lentiviral model, a robust reduction in ATXN3MUT protein levels is observed in the hemisphere expressing G3BP1 (ATXN3MUT+G3BP1: 0.285±0.04 vs. ATXN3MUT: 0.413±0.08, n=4, P=0.054) (Fig. 20d-e). No changes in ATXN2MUT or ATXN2MUT mRNA levels were observed between hemispheres (Fig. 20c, 20f). Expression of mutant ataxin-2 or mutant ataxin-3 in the striatum leads to loss of the neurogenic marker rs43,44.In accordance with the results above, G3BP1 expression led to the preservation of the neuronal marker DARPP-32 in both models compared to the control hemisphere (ATXN2MUT+G3BP1: 0.02 ± 0.0078, vs. ATXN2MUT: 0.08 ± 0.0078, n = 5, P = 0.001; ATXN3MUT+G3BP1: 0.19 ± 0.0291, vs. ATXN3MUT: 0.45 ± 0.0647, n = 7, P = 0.0072) (Fig. 6c, 6e, 6f, 6h). Taken together, these results indicate that G3BP1 expression in the striatum mediates neuroprotection and reduces the neuropathological features associated with mutant ataxin-2 and mutant ataxin-3 expression.

[0085] Overexpression of G3BP1 in wild-type mouse brain did not result in neuronal loss or astrogliosis Based on the previous results, the inventors of the present disclosure evaluated the impact of G3BP1 expression in the brain of wild-type animals. To this end, lentiviral particles encoding G3BP1 were injected into one hemisphere of the striatum of wild-type C57BL / 6 mice, and the contralateral hemisphere was injected with PBS as a control (Figure 7a). Four weeks after injection, the loss of the neuronal marker DARPP-32 in the G3BP1-injected hemisphere (Figure 7b) was significantly smaller compared to the PBS-injected control hemisphere (G3BP1: 0.003 ± 0.0014, vs. PBS: 0.01 ± 0.0011, n = 4, Student's t test, P = 0.035) (Figures 7b, 7c). Indeed, in G3BP1-injected animals, the lesion area was restricted to the injection site. In this line, we analyzed astrocyte activation via the GFAP marker, comparing G3BP1-injected hemispheres with control PBS-injected hemispheres (Fig. 7d). No differences in GFAP immunoreactivity were observed between the two hemispheres (Fig. 7e). Overall, these results point out that G3BP1 overexpression in normal brain does not appear to result in toxicity.

[0086] Restoring cerebellar G3BP1 levels alleviates behavioral deficits and neuropathological abnormalities in a transgenic mouse model of SCA3. PolyQ SCA is characterized by progressive neuronal loss and motor dysfunction. Thus, to mimic this phenotype, we used a transgenic mouse model expressing a truncated ataxin-3 mutant with 69 glutamines, characterized by severe motor dysfunction, neurodegeneration, and early onset of the disease. 23 This may also be a relevant polyQ model, given that only a small region of the ataxin-3 protein and a key glutamine pathway is required to cause pathology, as observed in other polyQ diseases. 23,48 We therefore investigated the effect of G3BP1 expression in this transgenic mouse model (Fig. 5g-i) in which the expression level of G3BP1 was reduced. To this end, at 4 weeks of age, animals were stereotactically injected into the cerebellum with a lentiviral vector encoding G3BP1. 49, control animals were injected with a lentiviral vector encoding GFP. A third group of non-injected animals was also used. Animals were then subjected to a battery of behavioral tests every 3 weeks until 9 weeks after injection. At this final time point, animals injected with GB3BP1 stayed on the rotating rotorod longer compared to control animals, showing enhanced motor impairment (G3BP1: 1.45 ± 0.0124, n = 7, vs. NI: 0.84 ± 0.1082, n = 7, P = 0.0254) (Figure 8a). In the same strain, 9 weeks after injection, in a swimming test in which animals must reach a safe platform across a pool, G3BP1-injected animals took less time to reach the platform compared to control animals (G3BP1: 0.55 ± 0.0974, n = 7, vs. NI: 0.99 ± 0.173, n = 7, P = 0.0476) (Figure 8b). Finally, in the footprint pattern test, in which animals paint their paws to cross a white sheet tunnel, animals injected with G3BP1 showed smaller footprint overlaps compared to control animals, suggesting an improved motor deficit (G3BP1: 1.06 ± 0.1081, n = 7; NI: 1.62 ± 0.1997, n = 7; P = 0.0297) (Figure 8c). Overall, these results indicate that G3BP1 expression in the cerebellum can ameliorate motor deficits. Neuropathologically, this mouse model is characterized by the formation of aggregates in Purkinje cells of the cerebellum, showing a significant reduction in the number of these cells and a strong disruption of the cerebellar layer structure. 23,50We therefore assessed the impact of G3BP1 expression on neuropathological abnormalities (Fig. 8d). In line with the observed improvement in motor deficits, we found that animals injected with G3BP1 had a significantly reduced number of pathological aggregates (HA tag) compared to controls (G3BP1: 63.12 ± 10.17, n = 6, vs. NI: 101.2 ± 15.29, n = 6, P = 0.0397) (Fig. 8e). As the expression of ATXN3MUT in our model is directed to Purkinje cells in the cerebellum, we also assessed their number using the calbindin marker. We found that animals injected with G3BP1 had a preserved Purkinje cell number compared to controls (G3BP1: 1.62 ± 0.2405, n = 6, vs. NI: 0.91 ± 0.1904, n = 6, P = 0.0437) (Fig. 8f). Importantly, in non-transduced lobes, no differences were observed between the experimental groups in terms of pathological aggregates (G3BP1: 44.45 ± 7.169, n = 6, vs. NI: 49.7 ± 9.385, n = 6) or cerebellar Purkinje cell numbers (G3BP1: 1 ± 0.1457, n = 6, vs. NI: 0.97 ± 0.1988 n = 6) (Figure 21). As these transgenic animals show a strong atrophy of the cerebellum, the thickness of the cerebellar layers was analyzed. The molecular layer thickness (II / III) of the transduced lobes was found to be significantly wider compared to the non-injected controls (G3BP1: 64.99 ± 3.189, n = 6, vs. NI: 56.03 ± 1.824, n = 6, P < 0.0118), whereas no differences were observed in non-transduced lobes (Figure 22). Overall, these results indicate that G3BP1 expression in the cerebellum significantly reduces motor behavioral deficits and decreases neuropathological abnormalities.

[0087] Consideration Proteins containing abnormally expanded polyQ tracts have been associated with the impairment of several cellular pathways that ultimately lead to cell death. The high tendency of mutant polyQ proteins to abnormally aggregate is directly involved or at least contributes to the exacerbation of certain toxic outcomes, acting crucially in the pathogenesis of polyQ. During the past decade, it has been hypothesized that abnormal protein aggregation, characteristic of several neurodegenerative diseases, not only exposes cells to stress but also impairs cellular stress response pathways. 51The formation of stress granules is one of the key roles in stress response, as they play a key role as mediators of protein synthesis. During the assembly of SGs, some important players, such as RBPs and mRNAs, are sequestered in the granules, preventing these components from integrating the translational machinery. 12,52 Furthermore, previous evidence has shown that SGs coexist with several protein aggregates, which are characteristic of various neurodegenerative diseases. 53 Therefore, the inventors of the present disclosure hypothesized that activation of the stress response through the formation of SGs could sequester mutant PolyQ proteins or promote translational arrest and reduce their expression. We found that chemical induction of SG assembly in Neuro2a cells expressing mutant ataxin-2 or mutant ataxin-3 led to a significant reduction in overall translation levels, but did not interfere with the expression levels of both ATXN2MUT and ATXN3MUT, nor did it appear to interfere with the aggregation of those proteins.

[0088] G3BP1 is a core component of SGs and an RBP that can induce SG biogenesis in the dephosphorylated state. 13 It has been reported that cellular stress induced by sodium arsenite reduces the constitutive phosphorylation state of G3BP1. 13,54 However, in recent years, this hypothesis has been challenged. 54However, it is unclear whether there is a correlation between sodium arsenite-mediated cellular stress induction and the phosphorylation / dephosphorylation state of G3BP1. To clarify this possible relationship, the inventors of this disclosure overexpressed G3BP1 in SCA2 patient-derived fibroblasts. We found that G3BP1 exhibited diffuse expression in cells, which is in contrast to what occurs when cells are treated with sodium arsenite. Upon sodium arsenite treatment, G3BP1 self-assembles in structures resembling SGs. Since G3BP1 function varies depending on its phosphorylation / dephosphorylation state, our next aim was to study the effects of G3BP1 overexpression in Neuro2a cells expressing ATXN2MUT e ATXN3MUT. We observed a decrease in the number of cells with mutant protein aggregates and the expression level of mutant polyQ proteins upon G3BP1 overexpression. We hypothesized that phosphorylated G3BP1 diffuses into cells and performs its catalytic activity on mutant polyQ proteins, whereas non-phosphorylated G3BP1 assembles into SG-like structures and switches its function.

[0089] To clarify the specificity of G3BP1 action on ATXN2MUT and ATXN3MUT levels and aggregates, this disclosure used Neuro2a cells, which have low levels of mouse endogenous G3BP1. It was observed that when ATXN2MUT and ATXN3MUT were expressed in this cell line, the number of cells with aggregates was maintained compared to the normal Neuro2a cell line. However, when co-expressing G3BP1 and the mutant proteins, the results are in line with what is observed in Neuro2a cells, i.e., a reduction in the number of cells with aggregates. These observations suggest that G3BP1 expression is involved in the reduction of the levels of mutant polyQ proteins and the number of aggregates.

[0090] Next, we investigated which domains of G3BP1 are involved in the molecular mechanism of its action. Thus, this study focused on the NTF2-like domain, which has been shown to be involved in nuclear transport through nuclear pores and to promote protein-protein interactions. 55In addition, we investigated the contribution of the RRM domain, which can interact with target RNA sequences and bind other proteins. 56 By expressing truncated constructs of G3BP1 with deletion of the NTF2 or RRM domain, a decrease in the number of cells with aggregates of both ATXN2MUT and ATXN3MUT and their expression levels was observed when the RRM domain was deleted. Conversely, no difference was found between the experimental conditions when NTF2 was deleted. This suggested that the NTF2 domain is essential for the action of G3BP1. Next, the inventors analyzed the effect of G3BP1 expression on the mRNA levels of ATXN2MUT and ATXN3MUT. These levels were found to be significantly decreased upon G3BP1 expression. The G3BP1 protein has been found to interact with ATXN3 RNA, 40 , which may account for the more robust results seen with ATXN3 mRNA compared to ATXN2. Previous studies have shown that phosphorylated G3BP1 translocates to the cell nucleus, where it likely exerts endoribonuclease activity. 17,33 As mentioned above, the NTF2-like domain of G3BP1 is very close to a critical phosphorylation site, serine 149, which is also thought to be linked to the endonuclease activity of G3BP1. 33 To assess the effect of G3BP1 phosphorylation, we performed site-directed mutagenesis in G3BP1 to switch serine 149 for alanine, thereby generating a phosphorylated death protein at the 149 aa site. Using this phosphorylated death construct, we found that expression of G3BP1 lost its effect on the number of cells harboring ATXN2MUT and ATXN3MUT aggregates, suggesting that phosphorylation of G3BP1 is crucial for its molecular function.

[0091] Next, the inventors of the present disclosure analyzed G3BP1 expression levels in the context of SCA2 and SCA3 patients and animal models. In postmortem samples of human brain tissue from SCA2, G3BP1 staining was found to be substantially reduced, suggesting low levels of expression. Accordingly, SCA2 and SCA3 patient-derived fibroblasts were also observed to have reduced levels of G3BP1 mRNA and protein. The results showed that the molecular pathological phenotypes observed in SCA2 and SCA3 are exacerbated due to the joint effect of polyQ mutant protein toxicity and low expression levels of G3BP1. Therefore, using different mouse models of these two diseases, the possibility of G3BP1 re-establishment in disease alleviation was investigated. Using SCA2 and SCA3 lentiviral mouse models, it was observed that injection of lentiviral particles encoding G3BP1 into the striatum led to the preservation of brain tissue (DARPP-32 staining) and a reduction in the number of aggregates. Furthermore, in a transgenic mouse model characterized by severe neurodegeneration and motor deficits, injection of lentiviral particles encoding G3BP1 into the cerebellum was found to reduce the number of aggregates and preserve the number of Purkinje cells. Importantly, expression of G3BP1 in the mouse cerebellum significantly improved overall motor skills, balance, and coordination.

[0092] The present disclosure surprisingly found that G3BP1 expression levels were decreased in both patient-derived fibroblasts and brain samples from SCA2 and SCA3 patients. Furthermore, it was shown that G3BP1 expression can reduce the expression of mutant ataxin-2 and ataxin-3. These results strongly support that the ability of G3BP1 to downregulate mutant ataxin-2 and ataxin-3 in SCA2 and SCA3 disease is impaired by reduced G3BP1 expression levels, leading to exacerbated phenotypes. In addition, it was also shown that the G3BP1 NTF2-like domain and ser 149 phosphorylation site are essential for alleviating mutant ataxin-2 and mutant ataxin-3 aggregation.

[0093] The results of the present disclosure strongly support that gene delivery of G3BP1 is efficient and safe in palliative SCA2 and SCA3 pathology and support G3BP1 as a novel therapeutic target not only for SCA2 and SCA3, but also for other PolyQ diseases.

[0094] Whenever used in this document, the term "comprising" is intended to indicate the presence of stated features, integers, steps, components, but does not exclude the presence or addition of one or more other features, integers, steps, components, or groups thereof.

[0095] The present disclosure is not limited to the described embodiments, and those skilled in the art envision many possibilities for modifying it. The above-described embodiments can be combined.

[0096] The following dependent claims further describe particular embodiments of the present disclosure.

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RasGAP-Associated Endoribonuclease G3BP: Selective RNA Degradation and Phosphorylation‐Dependent Localization. Mol. Cell. Biol. 21, 7747‐7760 (2001). 34. Kim, S. S. Y., Sze, L. & Lam, K. P. The stress granule protein G3BP1 binds viral dsRNA and RIG‐I to enhance interferon-β response. J. Biol. Chem. 294, 6430‐6438 (2019). 35. Lunde, B. M., Moore, C. & Varani, G. RNA-binding proteins: Modular design for efficient function. Nature Reviews Molecular Cell Biology 8, 479‐490 (2007). 36. Alam, U. & Kennedy, D. Rasputin a decade on and more promiscuous than ever? A review of G3BPs. Biochim. Biophys. Acta‐Mol. Cell Res. 1866, 360‐370 (2019). 37. Nagai, K., Oubridge, C., Ito, N., Avis, J. & Evans, P. The RNP domain: a sequence-specific RNA‐binding domain involved in processing and transport of RNA. Trends in Biochemical Sciences 20, 235‐240 (1995). 38. Vognsen, T., Moller, I. R. & Kristensen, O. Crystal structures of the human G3BP1 NTF2‐like domain visualize FxFG Nup repeat specificity. 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Claims

1. 1. An isolated or artificial nucleotide sequence encoding the protein G3BP1 for use in medicine or veterinary medicine, An isolated or artificial nucleotide sequence that is at least 95% identical to a sequence selected from the list consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and mixtures thereof.

2. 2. An isolated or artificial nucleotide sequence for use according to claim 1, which is identical to a sequence selected from the list consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and mixtures thereof.

3. 2. The isolated or artificial nucleotide sequence of claim 1 for use in the treatment of central and peripheral nervous system disorders.

4. 2. The isolated or artificial nucleotide sequence of claim 1 for use in the treatment of a neurodegenerative disease.

5. 2. The isolated or artificial nucleotide sequence according to claim 1 for use in the treatment of movement disorders, i.e. deficits in balance, motor coordination and / or motor performance.

6. 2. The isolated or artificial nucleotide sequence of claim 1 for use in the treatment of a polyglutamine disease.

7. 1. An isolated or artificial nucleotide sequence for use in the treatment of a polyglutamine disease, comprising:

2. The isolated or artificial nucleotide sequence of claim 1, wherein the polyglutamine disease is positively influenced by the control of protein aggregation.

8. 8. The isolated or artificial nucleotide sequence for use according to claim 7, wherein the control of protein aggregation is the control of protein aggregation caused by the expansion of polyglutamine segments of the affected protein.

9. 1. An isolated or artificial nucleotide sequence for use in the treatment of a polyglutamine disease, comprising:

2. The isolated or artificial nucleotide sequence for use according to claim 1, wherein the polyglutamine disease is selected from the group consisting of Huntington's disease (HD), spinal-bulbar muscular atrophy (SBMA), dentatorubral-pallidoluysian atrophy (DRPLA), and polyglutamine repeat spinocerebellar ataxia.

10. 10. The isolated or artificial nucleotide sequence for use according to claim 9, wherein the polyglutamine repeat spinocerebellar ataxia is selected from the group consisting of spinocerebellar ataxia type 1 (SCA1), spinocerebellar ataxia type 2 (SCA2), spinocerebellar ataxia type 3 (SCA3), spinocerebellar ataxia type 6 (SCA6), spinocerebellar ataxia type 7 (SCA7), and spinocerebellar ataxia type 17 (SCA17).

11. 2. The isolated or artificial nucleotide sequence for use according to claim 1, wherein the sequence is administered directly into the brain of a patient or into the spinal cord of a patient.

12. 2. The isolated or artificial nucleotide sequence for use according to claim 1, wherein the sequence is administered by intravascular, intravenous, intranasal, intracerebroventricular or intrathecal injection.

13. A vector or construct comprising the isolated or artificial nucleotide sequence of claim 1 for use in medicine.

14. 14. The vector for use according to claim 13, wherein the vector is selected from the group of adenovirus, lentivirus, retrovirus, herpesvirus and adeno-associated virus (AAV) vectors.

15. The vector for use according to claim 13, wherein the vector is a lentiviral vector.

16. The vector for use according to claim 13, wherein the vector is an AAV vector.

17. A host cell comprising the vector according to claim 13 for use in medicine.

18. A protein G3BP1 encoded by an isolated or artificial nucleotide sequence for use in medicine, comprising: The protein G3BP1, wherein the sequence is at least 95% identical to a sequence selected from the list consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and mixtures thereof.

19. A pharmaceutical composition for use in medicine or veterinary medicine, comprising a therapeutically effective amount of an isolated or artificial nucleotide sequence according to any one of claims 1 to 13, or a vector according to any one of claims 14 to 16, or a host cell according to claim 17, or a protein according to claim 18, or a combination thereof.

20. A kit for use in a pharmaceutical or veterinary medicine, comprising: A kit comprising an isolated or synthetic nucleotide sequence according to any one of claims 1 to 13, or a vector according to any one of claims 14 to 16, or a host cell according to claim 17, or a protein according to claim 18, or a combination thereof.

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