Patent Application
20240238385
The present disclosure relates to isolated or artificial nucleotide sequences encoding the GTPase-activating protein-binding protein 1 (G3BP1), for use in medicine, preferably in the treatment of polyglutamine diseases. Furthermore, the present invention is also related to a vector comprising such sequence, a host cell comprising such vector, a protein G3BP1, or a composition thereof, for use for use in medicine, preferably in the treatment of polyglutamine diseases.
Polyglutamine (PolyQ) diseases are a group of hereditary neurodegenerative diseases including Huntington's disease (HD), Spinal bulbar muscular atrophy (SBMA), Dentatorubral-pallidoluysian atrophy (DRPLA), and several spinocerebellar ataxias (SCA1, 2, 3, 6, 7, and 17). These diseases are characterized by abnormal expansions of the trinucleotide CAG in coding regions of each disease-associated gene, which encode for an expanded polyglutamine tract in the respective proteins. A central feature of these diseases is the aggregation of the mutant protein, which promotes aberrant interactions with other proteins and mRNAs, leading to the impairment of several cellular pathways and organelles1. Nevertheless, the complete picture of the molecular events leading to selective neurodegeneration of specific brain region is yet to be fully understood. Moreover, until now there are no therapies able to stop or delay the disease progression that culminates in the premature death of patients with PolyQ diseases.
SCA2 and SCA3 (or Machado-Joseph disease—MJD) are two of the most prevalent spinocerebellar ataxias, being both characterized by a neurodegenerative profile that mainly affects the cerebellum and the brain stem. SCA2 is caused by an abnormal mutation in the ATXN2 gene above 31-33 CAG repeats, resulting in an overexpanded ataxin-2 protein2. SCA3 is caused by an abnormal mutation above 44-45 CAG in the ATXN3 gene, causing the ataxin-3 protein to be abnormally expanded3,4. Both mutant ataxin-2 and ataxin-3, are prone to aggregate and form large inclusions capable of sequestering other proteins. Though large inclusions are often reported as hallmarks of the disease, whether they are directly leading to toxicity is still a matter of debate5-8.
The pathological aggregation of polyQ proteins and the abnormal interactions in which they engage can result in significant changes in the cellular stress response pathways 9,10. To cope with stress, cells display several mechanisms that promote survival, including the assembly of stress granules (SGs). These are transiently formed foci that act in the triage and regulation of RNA during stress periods11. Recently, SGs dysregulation has been suggested to underlie the pathogenesis of several diseases, including neurodegenerative disorders12. SGs are dynamic structures that can have different compositions depending on the type of stress and type of cell, although RNA binding proteins (RBPs) are their main components. One of these components, which is also a core nucleator and marker of SGs is the GTPase-activating protein-binding protein 1 (G3BP1)13,14. G3BP1 has an important role in mRNA stabilization, degradation, and in splicing modulation15,16,17. Structurally, 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 crucial for G3BP1 ability to bind mRNAs, whereas the latter is involved in the nuclear import of proteins through the pore complex15. Additionally, the phosphorylation of G3BP1 in its Ser-149 residue was referred to being important in SGs assembly, although recent studies do not support his hypothesis. While all these domains and catalytic site are important for the G3BP1 functions, the role of each one of them in specific steps of the RNA metabolism is yet to be elucidated13,14.
Despite the extensive efforts developed over the last years, polyQ diseases pathogenesis is not completely understood, neither exist therapeutic options exist to delay or stop the disease progression. Therefore, it is essential to identify new molecular targets implicated in the disease pathogenesis and to develop new therapeutic strategies for this group of disorders.
These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.
In the present invention it was investigated the involvement of G3BP1, a SG component in the SCA2 and SCA3 pathogenesis, and its suitability as a target for therapy. It was observed that G3BP1 overexpression led to a significant reduction in the number of cells with aggregates and in the levels of ataxin-2 and ataxin-3 proteins. The NTF2-like domain and Ser149 residue seems to be important in this mechanism of action of G3BP1. Moreover, it was found that G3BP1 levels are reduced in SCA2 and SCA3 patients' samples. Importantly, in SCA2 and SCA3 lentiviral mouse models, the knockdown of the G3bp1 levels increased the number of aggregates, highlighting an important functional role of this protein in the context of SCA2 and SCA3. On the contrary, the re-establishment of G3BP1 levels in a lentiviral mouse model of SCA2 and of SCA3 reduced neuropathological anomalies associated with the expression of mutant ataxin-2 or mutant ataxin-3, respectively. In the same line, G3BP1 expression was able to significantly reduce behaviour and neuropathological deficits in a transgenic mouse model. Altogether, the inventors had surprisingly identified G3BP1 as a relevant target for polyQ diseases, namely SCA2 and SCA3 and disclose therapeutic strategies for such diseases.
The inventors have shown that there is a reduction in G3BP1 levels in patients with a SCA2 and SCA3 disease. Based on this discovery, the inventors successfully studied the possibility to address the modulation of G3BP1 expression as a therapeutic strategy to counteract SCA2 and SCA3, by use of a vector encoding nucleic acid that expresses G3BP1 in the target cells.
It is therefore an object of the present invention to provide an isolated or artificial nucleotide sequence encoding the GTPase-activating protein-binding protein 1 (G3BP1), for use in medicine or veterinary, preferably in the treatment of polyglutamine diseases. Furthermore, the present invention is also related to a vector comprising such sequence, a host cell comprising such vector, a protein G3BP1, or a composition thereof, for use for use in medicine or veterinary, preferably in the treatment of polyglutamine diseases.
In an embodiment, the G3BP1 protein is the protein identified by the NCBI sequence reference: NP_005745.1), as encoded by the nucleotide sequence identified by the NCBI sequence reference GI: 10146 (GeneBank accession: NM_005754.3).
An aspect of the present disclosure relates to an isolated or artificial nucleotide sequence encoding the protein G3BP1, wherein the sequence is at least 95% identical to sequence selected from a list consisting of: SEQ. ID. 1; SEQ. ID. 2; SEQ. ID 3, SEQ. ID. 4; SEQ. ID 5; SEQ. ID 6; SEQ. ID 7 and mixtures thereof, for use in medicine or veterinary.
In an embodiment, the isolated or artificial nucleotide sequence for use in medicine or veterinary is identical to a sequence selected from a list consisting of: SEQ. ID. 1; SEQ. ID. 2; SEQ. ID 3, SEQ. ID; SEQ. ID 5; SEQ. ID 6; SEQ. ID 7, and mixtures thereof.
In an embodiment, the isolated or artificial nucleotide sequence may be used in the treatment of central and peripherical nervous system diseases
In another embodiment, the isolated or artificial nucleotide sequence may be used in the treatment of neurodegenerative diseases.
In another embodiment, the isolated or artificial nucleotide sequence may be used in the treatment of a movement disorder, namely lack of balance, motor coordination and/or motor performance.
In another embodiment, the isolated or artificial nucleotide sequence may be used in the treatment of a polyglutamine disease.
In another embodiment, the isolated or artificial nucleotide sequence may be used in the treatment of a polyglutamine disease wherein said diseases are positively influenced by the control of protein aggregation, wherein said control of protein aggregation is the control of protein aggregation caused by an expansion in the polyglutamine segment of the affected proteins.
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.
In another embodiment, the isolated or artificial nucleotide sequence may be used in the treatment of 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).
In another embodiment, the isolated or artificial nucleotide may be to be administered directly into the brain of the patient or into the spinal cord of the patient.
In another embodiment, the isolated or artificial nucleotide may be to be administered by intravascular, intravenous, intranasal, intraventricular or intrathecal injection.
Another aspect of the present disclosure relates to a vector or construct comprising an isolated or artificial nucleotide sequence as described above.
In an embodiment, the vector is selected from the group of adenovirus, lentivirus, retrovirus, herpesvirus and Adeno-Associated Virus (AAV) vector.
In another embodiment, the vector is a lentiviral vector.
Another aspect of the present disclosure relates to a host cell comprising the vector described above for use in medicine or veterinary.
Another aspect of the present disclosure relates to a protein G3BP1 encoded by an isolated or artificial nucleotide sequence, wherein the sequence is at least 95% identical to a sequence selected from a list consisting of: SEQ. ID. 1; SEQ. ID. 2; SEQ. ID 3, SEQ. ID; SEQ. ID 5; SEQ. ID 6; SEQ. ID 7, and mixtures thereof, for use in medicine or veterinary.
Another aspect of the present disclosure relates to a pharmaceutical composition for use in medicine or veterinary comprising a therapeutically effective amount of an isolated or artificial nucleotide sequence as described above, or a vector as described above, or a host cell as described above, or a protein as described above, or combinations thereof.
Another aspect of the present disclosure relates to a kit for use in medicine or veterinary comprising an isolated or synthetic nucleotide sequence as described above, or a vector as described above, or a host cell as described above, or a protein as described above, or combinations thereof
The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.
The present disclosure relates to an isolated or artificial nucleotide sequence encoding the GTPase-activating protein-binding protein 1 (G3BP1), for use in medicine, preferably in the treatment of polyglutamine diseases. Furthermore, the present invention is also related to a vector comprising such sequence, a host cell comprising such vector, a protein G3BP1, or a composition thereof, for use for use in medicine, preferably in the treatment of polyglutamine diseases.
In an embodiment, an isolated or artificial sequence encoding the protein G3BP1 can be selected from the list present in Table 1.
In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this invention. The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element, e.g., a plurality of elements. The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to”. The term “or” is used herein to mean, and is used interchangeably with, the term “and/or,” unless context clearly indicates otherwise. For example, “sense strand or antisense strand” is understood as “sense strand or antisense strand or sense strand and antisense strand.” The term “about” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as about 2 standard deviations from the mean. In certain embodiments, about means+10%. In certain embodiments, about means+5%. When about is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range. The term “at least” prior to 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 could logically be included, as clear from 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 of the numbers in the series or range.
In the context of the invention, the terms “treatment”, “treat” or “treating” are used herein to characterize a therapeutic method or process that is aimed at (1) slowing down or stopping the progression, aggravation, or deterioration of the symptoms of the disease state or condition to which such term applies; (2) alleviating or bringing about ameliorations of the symptoms of the disease state or condition to which such term applies; and/or (3) reversing or curing the disease state or condition to which such term applies.
As used herein, the term “subject” or “patient” refers to an animal, preferably to a mammal, even more preferably to a human, including adult and child. However, the term “subject” can also refer to non-human animals, in particular mammals such as mouse, and non-human primates.
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.
As used herein, the terms “coding sequence” or “a sequence which encodes a particular protein”, denotes a nucleic acid sequence which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide 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.
“G, “C”, “A,” “T,” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine, and uracil as a base, respectively.
In an embodiment, the invention describes an isolated or artificial sequence or a variant thereof for use in medicine.
The variants include, for instance, naturally-occurring variants due to allelic variations 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 the sequences SEQ. ID. 1-7, i.e., exhibit a nucleotide sequence identity of typically at least 90%, preferably at least 95%, more preferably at least 98%, more preferably at least 99% with one of the sequences SEQ. ID. 1-7.
Methods for the alignment of sequences for comparison are well known in the art, 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 the global (over the whole the sequence) 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 percent sequence identity and performs a statistical analysis of the similarity between the two sequences. The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI). Global percentages of similarity and identity may 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). Minor manual editing may be performed to optimise alignment between conserved motifs, as would be apparent to a person skilled in the art. The sequence identity values, which are indicated in the present subject matter as a percentage were determined over the entire amino acid sequence, using BLAST with the default parameters.
In an embodiment, the vector use according to the present invention is a non-viral vector. Typically, the non-viral vector may be a plasmid encoding G3BP1. This plasmid can be administered directly or through a liposome, an exosome or a nanoparticle.
Gene delivery viral vectors useful in the practice of the present invention can be constructed utilizing methodologies well known in the art of molecular biology. Typically, viral vectors carrying transgenes are assembled from polynucleotides encoding the transgene, suitable regulatory elements and elements necessary for production of viral proteins which mediate cell transduction.
The terms “gene transfer” or “gene delivery” refer to methods or systems for reliably inserting foreign DNA into host cells. Such methods can result in transient expression of non-integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g. episomes), or integration of transferred genetic material into the genomic DNA of host cells.
In an embodiment, examples of viral vector include adenovirus, lentivirus, retrovirus, herpes-virus and Adeno-Associated virus (AAV) vectors.
Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. 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 may be found for instance in WO95/14785, WO96/22378, U.S. Pat. Nos. 5,882,877, 6,013,516, 4,861,719, 5,278,056 and WO94/19478.
In a preferred embodiment, lentiviral vectors are employed.
Lentiviral vectors typically are generated by trans-complementation in packaging cells that are co-transfected with a plasmid containing the vector genome and the packaging constructs that encode only the proteins essential for lentiviral assembly and function. A self-inactivating (SIN) lentiviral vector can be generated by abolishing the intrinsic promo ter/enhancer activity of the HIV-1 LTR, which reduces the likelihood 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 to generate lentiviral vectors is to co-transfect cell lines (e.g., 293T human embryonic kidney cells) with a lentiviral vector plasmid and three packaging constructs encoding the viral Gag-Pol, Rev-Tat, and envelope (Env) proteins.
Methods of delivery, or administration, of viral vectors to neurons and/or astrocytes and/or oligodendrocytes and/or microglia include generally any method suitable for delivery vectors to said cells, directly or through hematopoietic cells transduction, such that at least a portion of cells of a selected synaptically connected cell population is transduced. The vector may be delivered to any cells of the central nervous system, cells of the peripheral nervous system, or both. Preferably, the vector is delivered to cells of the brain. Generally, the vector is delivered to the cells of the brain, including for example cells of brainstem (medulla, pons, and midbrain), cerebellum, susbtantia nigra, striatum (caudate nucleus and putamen), frontotemporal lobes, visual cortex, spinal cord or combinations thereof, or preferably any suitable subpopulation thereof.
Additional routes of administration may also comprise local application of the vector under direct visualization, e. g., superficial cortical application, intranasal application, or other nonstereotactic application.
The target cells of the vectors of the present invention are cells of the brain of a subject afflicted with PolyQ SCA, preferably neural cells. Preferably the subject is a human being, generally an adult but may be a child or an infant.
The present invention also encompasses delivering the vector to biological models of the disease. In that case, the biological model may be any mammal at any stage of development at the time of delivery, e. g., embryonic, foetal, infantile, juvenile or adult, preferably it is an adult. Furthermore, the target cells may be essentially from any source, especially nonhuman primates and mammals of the orders Rodenta (mice, rats, rabbit, hamsters), Carnivora (cats, dogs), and Arteriodactyla (cows, pigs, sheep, goats, horses) as well as any other non-human system (e. g. zebrafish model system).
Preferably, the method of the invention comprises intracerebral administration, through stereotaxic injections. However, other known delivery methods may also be adapted in accordance with the invention. For example, for a more widespread distribution of the vector across the brain, it may be injected into the cerebrospinal fluid, e. g., by lumbar puncture, cisterna magna or ventricular puncture. To direct the vector to the brain, it may be injected into the spinal cord or into the peripheral ganglia, or the flesh (subcutaneously or intramuscularly) of the body part of interest. In certain situations the vector can be administered via an intravascular approach. For example, the vector can be administered intra-arterially (carotid) in situations where the blood-brain barrier is disturbed. Moreover, for more global delivery, the vector can be administered during the “opening” of the blood-brain barrier achieved by infusion of hypertonic solutions including mannitol or ultra-sound local delivery.
The vectors used herein may be formulated in any suitable vehicle for delivery. For instance, they may be placed into a pharmaceutically acceptable suspension, solution or emulsion. Suitable mediums include saline and liposomal preparations. More specifically, pharmaceutically acceptable carriers may include sterile aqueous 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 and buffered media. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.
A colloidal dispersion system may also be used for targeted gene delivery. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes or exosomes.
The invention will be further illustrated by the following example. However, this example and the accompanying figures should not be interpreted in any way as limiting the scope of the present invention.
In an embodiment, plasmids encoding for human ataxin-3 contain 28 glutamines (pEGFP-C1-Ataxin3Q28; #22122; Addgene) or 84 glutamines (pEGFP-C1-Ataxin3Q84; #22123; Addgene) were a gift from Henry Paulson and both are fused with a GFP protein at the N-terminal18. Plasmids encoding for human ataxin-2 containing 22 glutamines (pEGFP-Ataxin2Q22) or 104 glutamines (pEGFP-Ataxin2Q104) were kindly provided by Prof. Stefan Pulst19. The LacZ gene was cloned in our laboratory under the control of a phosphoglycerate kinase promoter (PGK)20, and the GFP construct was cloned as previously described21. The plasmid encoding for human G3BP1 (SEQ. ID. 1) purchased from Source Bioscience, was cloned into a lentiviral vector backbone using the Gateway™ LR Clonase™ II Enzyme Mix, Invitrogen, according to the manufacturer instructions. The G3BP1-ΔNTF2 (G3BP1 deleted at the site 11-133) and G3BP1-ΔRRM (G3BP1 deleted at the site 340-415) constructs were synthesized from GeneScript and cloned into the vector pcDNA3.1+N-MYC. A validated shRNA targeting mouse G3bp1 (#MSH031039-LVRU6MP-b) and a shRNA scramble, as control (with no known target, #CSHCTR001-LVRU6MP) were acquired from GeneCopoeia (USA).
In an embodiment, the plasmid encoding for human G3BP1 (one of the sequences SEQ. ID. 1 to 7) were cloned into a self-inactivating lentiviral vector under the control of PGK promoter using the Gateway™ LR Clonase™ II Enzyme Mix, Invitrogen, according to the manufacturer instructions. The lentiviral vectors were produced in HEK (human embryonic kidney) 293T cells using a four-plasmid system described previously25. The viral productions were quantified using a RetroTek HIV-1 p24 Antigen Enzyme-Linked Immunoabsorbent Assay (ELISA) (ZeptoMetrix), according to manufacturer's indications.
In an embodiment, sited-directed mutagenesis was performed using NZY Mutagenesis kit (NZYTech) according to manufacturer's indications. In the human variant of G3BP1 (GeneBank accession: DQ893058.2), a serine was changed by an alanine or an aspartate at the site 149 to generate a G3BP1 phospho-dead mutant (G3BP1_S149A) or a G3BP1-phosphomimic mutant (G3BP1_S149D), respectively. The pair of primers used to induce the substitution S149A were: SEQ. ID. 8: 5′-CT GAG CCT CAG GAG GAG GCT GAA GAA GAA GTA GAG-3′ and SEQ. ID. 9: 5′-CT CTA CTT CTT CTT CAG CCT CCT CCT GAG GCT CAG-3′. The pair of primes used to induce the substitution S149D were: SEQ. ID. 10: 5′-CT GAG CCT CAG GAG GAG GAT GAA GAA GAA GTA GAG-3′ and SEQ. ID. 11: 5′-CTC TAC TTC TTC TTC ATC CTC CTC CTG AGG CTC AG-3′. The mutations S149A and S149D were confirmed by DNA sequencing (Eurofins Genomics).
In an embodiment, mouse neuroblastoma cell line (Neuro2a cells) acquired from the American Type Culture Collection cell biology bank (CCL-131) were cultured in Dulbecco's modified Eagle medium (DMEM), supplemented with 10% (v/v) foetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin. Cells were seeded onto 12- or 6-multiwell plates. After 24 h of growth, cells were transfected using polyethylenimine reagent (PEI; PEI MAX Polysciences, Inc.) following the manufacturer's instructions, with a concentration of 0.5-1 μg of DNA per well. For SGs induction experiments, cells were treated with sodium arsenite (SA, Sigma Aldrich 10 μg/mL) to a final concentration of 0.05 M, 1 h before harvest.
In an embodiment, patients fibroblasts from SCA2, SCA3, and healthy individuals were obtained from Coriell Institute or kindly provided by collaborators22, being fully characterized for CAG expansions: 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). Fibroblast cells were kept in culture in Dulbecco's modified Eagle medium (DMEM), supplemented with 15% (v/v) foetal 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.
In an embodiment, it was used a method that allows the monitoring and quantification of global protein synthesis based on the incorporation of puromycin during translation. N2a cells were plated into multiwell plates and transfected with lacZ or G3BP1. Twenty-four hours post-transfection the cells were incubated with 10 mg/ml of puromycin (Sigma) for 15 min, and after collected for western blot processing. As a positive control for the translation inhibition, some cells were incubated with 10 mM of cycloheximide (CHX, Sigma) for 15 min, and then incubated with 10 mg/ml of puromycin (Sigma) for an additional 15 min. For the stress granules condition, the cells were treated for 1 h with 0.05M sodium arsenite and then incubated with 10 mg/ml of puromycin (Sigma) for an additional 15 min. Additional controls of non-treated cells were also used.
In an embodiment, post-mortem striatum and cerebellum brain tissue from clinically and genetically confirmed SCA2 patients were obtained from the NIH NeuroBioBank (USA). Control striatum and cerebellum tissues from healthy individuals, without neurological conditions diagnosed were obtained from NIH NeuroBioBank (USA). Tissues preserved in 4% PFA solution, were dehydrated in a 30% sucrose/PBS for 48 h, cryoprotected at −80° C. degrees, dissected in 40 μm slices using a cryostat (Cryostar NX50, ThermoFisher Scientific) and stored in free floating PBS/sodium azide solution at 4° C.
In an embodiment, adult C57BL/6 J wild-type animals, and transgenic SCA3 mice23, breed in in the animal facility of the Universidade do Algarve, were used. The animals were maintained in a temperature-controlled room on a 12 h light-12 h fark cycle. Food and water were dispensed ad libitum. All experiments were carried out according to the European Community Council directive (86/609/EEC) for the care and use of laboratory animals. The researchers received certified training (FELASA course) and approval to perform the experiments from the Portuguese authorities (Direcção Geral de Alimentação e Veterinária) in the project Neuropath (421/2019).
In an embodiment, the cDNA encoding for human G3BP1, GFP, ATXN2MUT, and for ATXN3MUT was cloned in a self-inactivating lentiviral vector under the control of PGK promoter, as described previously24. The lentiviral vectors were produced in HEK (human embryonic kidney) 293T cells using a four-plasmid system described previously25. The viral productions were quantified using a RetroTek HIV-1 p24 Antigen Enzyme-Linked Immunoabsorbent Assay (ELISA) (ZeptoMetrix), according to manufacturer's indications.
In an embodiment, for the stereotaxic injection of lentiviral vectors, concentrated viral stocks were thawed on ice and homogenized. The animals were anesthetized through intraperitoneal injection (IP) of a mixture of ketamine (75 mg/kg, Nimatek, Dechra) with medetomidine (0.75 mg/kg, DOMTOR®, Esteve). For the SCA2 lentiviral mouse model, mice (10-12 weeks old) were injected with lentiviral particles encoding for human ATXN2MUT containing 82 glutamines or encoding for ATXN2MUT and G3BP1 at the left and right hemispheres of striatum, respectively, according to the following brain coordinates relative to bregma: Antero-Posterior (+0.6), Medial-Lateral (+/−1.8), Dorsal-Ventral (−3.3)26. A concentration of 400 ng p24/μl of lentivirus were injected at a rate of 0.20 μl/min. For the SCA3 lentiviral mice, viral particles encoding for human ATXN3MUT containing 72 glutamines or encoding for ATXN3MUT and G3BP1, were injected into mouse striatum (left hemisphere and right hemisphere, respectively) at 400 ng of p24/ml, using the same coordinates described above. To perform safety assays, wild-type C57/BL6 mice (10-12 weeks old) were injected into the striatum with lentiviral particles encoding for G3BP1 at a concentration of 400 ng p24/μl of lentivirus, using the same coordinates described above. For the transgenic animals, lentiviral particles encoding for G3BP1 or GFP, as respective control, were injected into mice cerebella (4 weeks old), at a concentration of 800 ng p24/μl of lentivirus at the coordinates: −1.6 mm rostral to lambda, 0.0 mm midline, and −1.0 mm ventral to the skull surface, with the mouth bar set at −3.321. For the G3bp1 silencing studies in SCA2, wild-type C57/BL6 mice (10-12 weeks old) were injected in the striatum with lentiviral particles encoding for human ATXN2MUT containing 82 glutamines and with lentiviral particles encoding for an shRNA scramble, whereas in the contralateral hemisphere the animals were injected with lentiviral particles encoding for human ATXN2MUT containing 82 glutamines and with lentiviral particles encoding for an shRNA targeting mouse G3bp1. For SCA3, the procedure was similar but using lentiviral particles encoding for human ATXN3MUT containing 72 glutamines. The lentiviral particles were injected at a concentration of 400 ng p24/μl of lentivirus, using the same coordinates described above. All stereotaxic injections were performed by means of an automatic injector (Stoelting Co.) using a 34-gauge blunt-tip needle linked to a Hamilton syringe. Mice were sacrificed for posterior analysis, a few weeks after surgery, 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.
In an embodiment, the transgenic mice were subjected to several motor behaviour tests starting before the stereotaxic injection (4 weeks of age), every 3 weeks until 9 weeks post-injection. Motor and gait coordination were accessed by rotarod and footprint tests in a blind fashion way following the same procedure described before21. In the footprint test analysis, steps taken by mice at the beginning and at the end of the walking test are not included and not considered for the measures. Swimming performance was assessed by placing mice at one end of a rectangular tank (100×10.5×20 cm), filled with water at room temperature. Mice freely swam for 1 m until they reached a platform and the time taken to transverse the tank was recorded. Mice performed the trial three times, with an interval of 15-20 minutes per trial. The mean of the time taken to cross the tank in the tree trials was used for statistical analysis.
In an embodiment, animals were sacrificed by sodium pentobarbital overdose and either transcardially perfused with 0.1M phosphate buffer solution and a 4% paraformaldehyde fixative solution (Sigma Aldrich) for immunohistochemical assays or had cervical dislocation and striatal punches of the brains, using a Harris Core pen with 2.5 mm diameter (Ted Pella Inc.), for qPCR and western blot analysis. The brains and the striatal punches collected were post-fixed in 4% paraformaldehyde for 24 h, dehydrated in a 30% sucrose/0.1M phosphate buffer solution (PBS) for 48 h and cryoprotected at −80° C. Sagittal or coronal brain sections of 30 μm and 25 μm, respectively, were obtained using the cryostat-microtome model CryoStar NX50 (Thermofisher). For preservation, brain sections were stored at 4° C., free-floating in 0.02% (w/v) sodium azide PBS
In an embodiment, to stain brain sections with cresyl violet, they were mounted in gelatin-coated microscope slides. Brain sections were sequentially submersed in water, ethanol 96% (v/v), ethanol 100% (v/v), xylene, ethanol 75% (v/v) and the 0.1% (w/v) cresyl violet solution. To wash slices, brain sections were sequentially submersed in water, ethanol 75% (v/v), ethanol 96% (v/v), ethanol 100% (v/v) and xylene. Finally, brain sections were mounted with Eukitt (Sigma-Aldrich). Images were acquired with 10× objective in a Zeiss Axio Imager Z2.
In an embodiment, for the immunocytochemical procedure, cells were fixed using 4% paraformaldehyde (PFA) fixative solution for 20 min and washed with 0.1 M phosphate buffer solution (PBS). Samples were then incubated in PBS containing 0.1% Triton™ X-100 for 10 min. Blocking in PSB with 1% of bovine serum albumin (Sigma) was performed for 30 min. Samples were incubated with the primary antibody overnight in the proper dilution at 4° C. and with the secondary antibody (1:200) for 2 h at room temperature. The secondary antibody was coupled to a fluorophore (Alexa Fluor®, Invitrogen). Finally, the coverslips were mounted on microscope slides using Fluoromount-G mounting media with DAPI (Invitrogen).
In an embodiment, the immunohistochemical procedure, for light imaging, started with the incubation of brain sections in phenylhydrazine diluted in phosphate buffer solution (1:1000; 15 min, 37° C.). For the human brain sections an additional step with a Tris-buffered saline pH 9 antigen retrieval method (30 min, 95° C.) was performed. Brain sections went through blocking in 10% normal goat serum in 0.1% Triton™ X phosphate-buffered solution (1 h, room temperature) and incubation with the respective primary (overnight at 4° C.) and secondary biotinylated antibodies (2 h at room-temperature) diluted in blocking solution, followed a reaction with the Vectastain elite avidin-biotin-peroxidase kit and by 3,3′-diaminobenzidine substrate (both from Vector Laboratories). Then, the sections were assembled over microscope slides, dehydrated in increasing degree ethanol solutions (75, 96 and 100%) and xylene, and finally cover slipped using mounting medium Eukitt (O. Kindler GmbH & CO). For fluorescence immunohistochemistry procedures, brain sections were incubated in the above-described blocking solution, followed by primary and secondary antibodies incubation. Brain sections were mounted in microscope slides with Fluoromount-G Mounting medium with 41, 6-Diamidino-2-Phenylindole, (DAPI) (Invitrogen).
In an embodiment, for 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); mouse β-Gal (14B7) (1:500, ref. 2372, Cell Signaling Technology).
In an embodiment, immunocytochemistry images were acquired in a Zeiss Axio Imager Z2 for quantification and in a Zeiss LSM710 confocal microscope for representative images. Quantitative analysis was blindly performed by counting the number of cells with aggregates within 100 transfected cells, using the 40x or 63x objective for each condition in each independent experiment. Immunohistochemistry images from the lentiviral mouse models were acquired with 20× objective in a Zeiss Axio Imager Z2 and Axio Scan.Z1 Slide Scanner microscopes. For quantification of ataxin-2 aggregates and DARP-32 staining loss, 18 coronal sections per animal were analyzed in ZEN lite software (Zeiss), so that a complete rostrocaudal picture of the striatum was obtained. Ataxin-2 inclusions were manually counted in all animals. DARP-32 neuronal lesion area was manually measured for all animals, allowing quantification of depleted volume according to the formula: volume=d*(a1+a2+a3), where d is the distance between serial sections (200 μm) and a1+a2+a3 are depleted areas for each individual section. Immunohistochemistry images from the transgenic mouse animals were acquired 8 sagittal sections, spanning 280 μm between them, of the entire cerebellum, stained with anti-HA, anti-Calbindin and DAPI were acquired with a Zeiss Axio Imager Z2 microscope using a 20x objective. For each section, the number of cells with HA aggregates and Purkinje cells were blindly counted in all cerebellar lobules using an image analysis software (ZEN 2.1 lite, Zeiss).
In an embodiment, samples were either lysed in 10x RIPA solution (Merck Millipore) if cell extracts or homogenized in a urea/DTT solution if mouse striatal punches, both containing a cocktail of protease inhibitors (Roche), followed by an ultrasound sonication of 30 sec ON, 30 sec OFF, 5 cycles (Bioruptor Pico). Protein concentration levels were determined using Pierce™ BCA Protein Assay Kit (Thermo Scientific) for cell lysates and NZYBradford reagent (Nzytech) for mouse samples. Protein extracts were resolved in SDS-polyacrylamide gels (7.5% and 12%), followed by protein transfer to PVDF membrane (Merck Millipore), membrane blocking in TBS-T with 3% of BSA or 5% of milk, and antibody probing overnight at 4° C. for primary and 2 h at room temperature for secondary. 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-β-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). Membranes were resolved using Enhanced Chemiluminescence (GE Healthcare) and scanned with a ChemiDoc™ XRS+ (Bio-Rad). Optical densiometric analysis was carried out using Image J software.
In an embodiment, total RNA from mouse striatal punches started by Trizol (Invitrogen) tissue dissociation and RNA/DNA/protein chloroform separation. Then, both mouse and cell samples were extracted with NZY Total RNA Isolation kit (Nzytech). RNA concentration and purity were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). cDNA molecules of 1 μg of RNA were produced using iScript cDNA synthesis kit (Bio-Rad) according to manufacturer recommendations. Quantitative RT-qPCR was performed with the SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad), using home-made primers for gene of interest and for the human GAPDH housekeeping gene as a control and performed in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). mRNA expression levels relative to mRNA gene control were determined using amplification values. The following primers were used: human ATXN2 (QT01852480) and human ATXN3 (QT00094927) from QuantiTect Primer Assays, Qiagen. Human G3BP1 (Forward SEQ. ID. 12: 5′-GAA ATC CAA GAG GAA AAG CC-3′; Reverse SEQ. ID. 13: 5′-CCC AAG AAA ATG TCC TCA AG), human GAPDH (Forward SEQ. ID. 14: 5′-ACA GTT GCC ATG TAG ACC-3′; Reverse SEQ. ID. 15: 5′-TTG AGC ACA GGG TAC TTT A-3′) and mouse Hprt (Forward SEQ. ID. 16: 5′-AGG GAT TTG AAT CAC GTT TG-3′; Reverse SEQ. ID. 17: 5′-TTT ACT GGC AAC ATC AAC AG-3′) from KiCqStart Pre-designed Primers, Sigma-Aldrich.
In an embodiment, statistical analysis was performed using either Student's t-test or one-way ANOVA complemented with Bonferroni multiple comparisons test, resorting to GraphPad software (La Jolla).
Stress Granules Assembly does not Alter the Levels of ATXN2 and ATXN3 Proteins.
SGs are cellular foci formed in response to stress in which mRNAs, translation factors, and RBPs coalesce together to prevent cellular damage27,28. Therefore, the inventors of the present disclosure investigated the impact of SGs assembly in ATXN2 and ATXN3 proteins dynamics, both in pathological (ATXN2MUT and ATXN3MUT) and non-pathological forms (ATXN2WT and ATXN3WT). For that, 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), SGs assembly was pharmacologically induced using sodium arsenite (
G3BP1 Overexpression Reduces the Number of Cells with Aggregates and the Levels of ATXN2 and ATXN3 Proteins
SGs assembly can also be induced by overexpression of its core components13,31, including G3BP1, which is an RBP able of both mRNA stabilization and degradation15. However, in the present disclosure it was observed that in Neuro2a G3BP1 overexpression alone is less effective in inducing SGs formation, than when combining it with a sodium arsenite stimulus (
G3BP1 is an RBP with several molecular and biological functions, including mRNA binding, DNA binding32, helicase, and has important functions in immune response34. Overall, RBPs, including G3BP1, interact with mRNAs through specific RNA-binding domains35,36. The RNA recognition motif (RRM) of G3BP1 is known for interacting with target RNA sequences37. G3BP1 also harbors a NTF2-like domain that is involved in the nuclear shuttling of proteins through the nuclear pore complex38, facilitates protein-protein interactions39, mediates G3BP1 dimerization, and is important in SGs formation13. Therefore, to better understand G3BP1 action on mutant ataxin-2 and ataxin-3 aggregation and levels, the inventors of the present disclosure developed two different forms of the protein, one with the deletion of the NTF2-like domain (G3BP1-ΔNTF2) and the other with the deletion of the RRM domain (G3BP1-ΔRRM) (
In G3BP1 protein, the NTF2-like domain is closely located to a phosphorylation site (Ser-149), which seems to have an important functional role17,36. The G3BP1-ΔRRM was able to reduce the levels and aggregation of ATXN2MUT and ATXN3MUT, however to a lesser extent than the full length G3BP1. Therefore, the inventors of the present disclosure aimed to investigated the importance of Ser149 in the functional role of G3BP1. For that, it was developed two phosphomutants of G3BP1, a phosphomimetic S149D and a nonphosphorylatable S149A (
G3BP1 mRNA and protein levels are reduced in SCA2 and SCA3, whereas silencing it increases aggregation in the mouse brain
Previous studies report that mutant polyQ proteins can dysregulate the expression of several genes1,41. In fact, the inventors of the present disclosure showed that the expression of mutant ataxin-3 drives an abnormal reduction of wild-type ataxin-2 levels42. In this line, it was then analyzed the levels of G3BP1 in samples from SCA2 and SCA3 patients and disease models. In post-mortem brain samples of SCA2 patients it was detected a reduction in the immunodetection of G3BP1, comparing with healthy individuals, both in striatum and cerebellum (
The expression of ATXN2MUT and ATXN3MUT, mediated by lentiviral vectors leads to the formation of intraneuronal aggregates and to the loss of neuronal markers43,44, which are neuropathological signs also found in post-mortem human tissue45-47. Thus, the inventors of the present disclosure investigated whether restoring G3BP1 levels improve neuropathological abnormalities induced by ATXN2MUT and ATXN3MUT in vivo. For that lentiviral vectors encoding ATXN2MUT (or ATXN3MUT) and human G3BP1 were co-expressed in one hemisphere of the striatum and, as a control, in the contralateral hemisphere, lentiviral vectors encoding ATXN2MUT (or ATXN3MUT) were injected (
Based on the previous results, the inventors of the present disclosure evaluated the impact of G3BP1 expression in the brain of wild-type animals. For that, lentiviral particles encoding G3BP1 were injected in one hemisphere of the striatum of wild-type C57BL/6 mice, while in the contralateral hemisphere was injected with PBS, as control (
PolyQ SCAs are characterized by a progressive neuronal loss and motor dysfunctionality. Thus, to mimic this phenotype, in the present disclosure a transgenic mouse model expressing a truncated form of mutant ataxin-3 with 69 glutamines was used and characterized by a severe motor dysfunctions, neurodegeneration and early onset23. This can also be a relevant polyQ model, considering that only contains a small region of the ataxin-3 protein, and a significant tract of glutamines, causing pathology, as observed in other polyQ diseases23,48. Therefore, it was then investigated the impact of G3BP1 expression in this transgenic mouse model, which has reduced levels of G3BP1 (
Proteins containing abnormally expanded polyQ tracts have been implicated with the impairment of several cellular pathways, which ultimately lead to cellular dead. The high propensity of the mutant polyQ proteins to aberrantly aggregate are either directly involved or at least contribute to aggravate particular toxic outcomes, acting decisively in the polyQ pathogenesis. In the last decade it has been hypothesized that the abnormal protein aggregation, characteristic of several neurodegenerative disorders, not only subjects cells to stress, but can also impair cellular stress-response pathways51. The formation of stress granules is one important player in stress response, as they play an important role as mediator of protein synthesis. During SGs assembly, several key players, such as RBPs and mRNAs are sequestered into the granule preventing these components from integrating the translational machineryl2,52. Previous evidence has also shown that SGs co-localize with several protein aggregates, which are characteristic of different neurodegenerative diseases53. Therefore, the inventors of the present disclosure hypothesized that an activation of the stress response through the formation of SGs could either sequestrate mutant polyQ proteins, or promote a translational arrest, decreasing its expression. It was found that chemically inducing SGs assembly, in Neuro2a cells expressing either mutant ataxin-2 our mutant ataxin-3, leads to a significant decrease in global translation levels, however it seems not to interfere with the expression levels of both ATXN2MUT and ATXN3MUT, nor with the aggregation of those proteins.
G3BP1 is an RBP, a core component of SGs and in its dephosphorylated state can induce SGs formation13. It has been reported that cellular stress induction by sodium arsenite, reduces the constitutive phosphorylation state of G3BP113,54. However, in recent years, this hypothesis was challenged54, and it is not clear if there is a correlation between cellular stress induction through sodium arsenite and phosphorylation/dephosphorylation status of G3BP1. To clarify this possible link, the inventors of the present disclosure overexpressed G3BP1 in SCA2 patients-derived fibroblasts. It was found that G3BP1 shows a diffuse expression within the cell, contrasting to what happens when we treat the cells with sodium arsenite. Upon sodium arsenite treatment, G3BP1 self-assembles in structures resembling SGs. As G3BP1 functions vary depending on its phosphorylation/dephosphorylation state, the next aim was to study the impact of G3BP1 overexpression in Neuro2a cells expressing ATXN2MUT e ATXN3MUT. Upon overexpression of G3BP1 it was observed a reduction in the number of cells with mutant protein aggregates and in the expression levels of mutant polyQ proteins. It was hypothesized that, while the phosphorylated G3BP1, diffusely spread in the cells, performs its catalytic activity on the mutant polyQ proteins, the non-phosphorylated G3BP1 assembles in SGs-like structures, switching its functions.
To clarify the specificity of G3BP1 action on ATXN2MUT and ATXN3MUT levels and aggregates, in the present disclosure Neuro2a cells with low levels of mouse endogenous G3bp1 were used. It was observed that when ATXN2MUT and ATXN3MUT are expressed in this cell line, the number of cells with aggregates is maintained, comparing to a normal Neuro2a cell line. However, when co-express G3BP1 and the mutant proteins are co expressed, the results are in line to what is observed in the in Neuro2a cells, i.e., a decrease in the number of cells with aggregates. These observations lead to suggest that is G3BP1 expression the responsible for the decrease of the levels and number of aggregates of mutant polyQ proteins.
Next, the inventors investigated which domains of G3BP1 could be implicated in its molecular mechanisms of action. Thus, the study was focused in the NTF2-like domain, which is involved in the nuclear transport via nuclear pores and has been shown to facilitate protein-protein interactions55. Additionally, it was also investigated the contribution of the RRM domain, which interacts with target RNA sequences and can also bind with other proteins56. By expressing truncated constructs of G3BP1 with deletions of NTF2 or RRM domains, it was observed a reduction in the number of cells with aggregates of both ATXN2MUT and ATXN3MUT and their expression levels, when the RRM domain is deleted. Opposingly, no differences were found among the experimental conditions when NTF2 is deleted. This led to suggest that NTF2 domain could be essential for G3BP1 action. The inventors next went to analyze the impact of G3BP1 expression on the mRNA levels of ATXN2MUT and ATXN3MUT. It was found that those levels were significantly decreased upon G3BP1 expression. G3BP1 protein was found to interact with ATXN3 RNA40, which could be the cause for the more robust results found in ATXN3 mRNA, comparing with ATXN2. Previous studies demonstrated that phosphorylated G3BP1 translocates to the cellular nucleus, probably to perform its endoribonuclease activityl7,33. As mentioned before, the NTF2-like domain of G3BP1 is very close to an important phosphorylation site, serine 149. This phosphorylation site is also believed to be connected to the endonuclease activity of G3BP133. To evaluate the impact of G3BP1 phosphorylation it was performed a sited-directed mutagenesis in G3BP1, switching the serine149 for an alanine, therefore generating a phospho-dead protein at the 149 aa site. Using this phospho-dead construct it was found that the expression of G3BP1 lost its impact in the number of cells with aggregates of ATXN2MUT and ATXN3MUT, leading us to suggest that G3BP1 phosphorylation is crucial for its molecular functions.
Next, the inventors of the present disclosure analyzed G3BP1 expression levels in the context of SCA2 and SCA3 patients and animal models. It was found that in postmortem samples of human brain tissue from SCA2, G3BP1 staining was substantially decreased, suggesting low levels of expression. Accordingly, it was also observed that SCA2 and SCA3 patient-derived fibroblasts have reduced levels of G3BP1 mRNA and protein. The results showed that molecular pathologic phenotype observed in SCA2 and SCA3 is exacerbated due to the joint effect of the polyQ mutant proteins toxicity and the lower expression levels of G3BP1. Therefore, it was then investigated the potential of G3BP1 re-establishment in disease mitigation, using different mice models of these two diseases. Using SCA2 and SCA3 lentiviral mouse models, it was observed that the injection into the striatum of lentiviral particles encoding for G3BP1 lead to the preservation of brain tissue (DARPP-32 staining) and to a decrease in the number of aggregates. Moreover, in a transgenic mouse model, characterized by severe neurodegeneration and motor deficits, it was found that the injection in the cerebellum of lentiviral particles encoding for G3BP1, reduces the number of aggregates and preserves the number of Purkinje cells. Importantly, G3BP1 expression in mice cerebella significantly improved the overall motor performance, balance, and coordination.
With the present disclosure it was surprisingly found that G3BP1 expression levels are decreased in both patient-derived fibroblast and brain sample of SCA2 and SCA3 affected individuals. Additionally, it was shown that G3BP1 expression can decrease the expression of mutant ataxin-2 and ataxin-3. These results strongly support that, in SCA2 and SCA3 disease, the ability of G3BP1 to downregulate the mutant ataxin-2 and ataxin-3 is impaired, due to G3BP1 decreased expression levels, leading to an exacerbation of the phenotype. Additionally, it was also shown that the G3BP1 NTF2-like domain and the ser 149 phosphorylation site, are essential to mitigate mutant ataxin-2 and mutant ataxin-3 aggregation.
The results of the present disclosure strongly support that gene delivery of G3BP1 is efficient and safe in the mitigation SCA2 and SCA3 pathology, supporting G3BP1 as a novel therapeutic target, not only for SCA2 and SCA3, but to other polyQ diseases.
The term “comprising” whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.
The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof. The above described embodiments are combinable.
The following dependent claims further set out particular embodiments of the disclosure.
| Number | Date | Country | Kind |
|---|---|---|---|
| 117230 | May 2021 | PT | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2022/054493 | 5/13/2022 | WO |
