An agent that increaes NATO3 expression and / or activity for use in the treatment of neurological disorders

Abstract

The present invention relates to an agent that increases Nato3 expression and / or activity for use in preventing or treating a neurological disorder and related methods and uses. The present invention also relates to an expression construct or viral vector encoding an agent that increases Nato3 expression and / or activity and a host cell comprising said vector. The present invention also relates to a dopaminergic neuron or a dopaminergic progenitor comprising an expression construct encoding an agent that increases Nato3 expression and / or activity and a method of screening for a compound that improves dopaminergic function in a dopaminergic neuron.

Description

[0001] TREATMENT OF NEUROLOGICAL DISORDERS Field of the Invention The present invention relates to an agent that increases Nato3 expression and / or activity for use in preventing or treating a neurological disorder and related methods and uses. The present invention also relates to an expression construct or viral vector encoding an agent that increases Nato3 expression and / or activity and a host cell comprising said vector. The present invention also relates to a dopaminergic neuron or a dopaminergic progenitor comprising an expression construct encoding an agent that increases Nato3 expression and / or activity and a method of screening for a compound that improves dopaminergic function in a dopaminergic neuron. Background to the Invention Neurological disorders such as Parkinson’s disease (PD) involve impairment of dopaminergic (DA) neurons. Nato3 ablation in postmitotic DA neurons has been shown to lead to impairments in mitochondrial health and the autophagy pathway in mice. Furthermore, overexpression of Nato3 homolog, Fer2, in flies prevents the demise of DA neurons in fly PD models (Miozzo et al., 2022). Existing treatments for PD include dopamine replacement therapy and deep brain stimulation to improve motor function but do not address underlying impairment. There is a need to provide further therapies for neurological disorders such as PD. Summary of the Invention The present invention relates to prevention or treatment of a neurological disorder by use of an agent that increases Nato3 expression and / or activity. The inventors surprisingly found that increasing Nato3 expression (and thus Nato3 activity) in human DA neurons derived from PD patients was able to reverse various impairments characteristic of PD. The ability to successfully restore dopaminergic function in DA neurons from PD patients by provision of increased Nato3 expression / activity was unexpected from previous animal / fly studies, which only illustrated effects of abrogating Nato3 activity in other organisms. Furthermore, the inventors found that viral infection of human DA neurons was an effective strategy for overexpression of Nato3. The inventors’ findings suggest that increasing Nato3 expression and / or activity (including in cells derived from human patients) may be an effective therapeutic strategy for PD and other neurological disorders. The present invention accordingly provides an agent that increases Nato3 expression and / or activity for use in a method for preventing or treating a neurological disorder. The invention further provides a method of preventing or treating a neurological disorder or increasing dopaminergic function comprising administering an agent that increases Nato3 expression and / or activity to a patient. The invention additionally provides use of an agent that increases Nato3 expression and / or activity in the manufacture of a medicament for preventing or treating a neurological disorder. The invention also provides an expression construct comprising a promoter operably linked to a nucleic acid sequence encoding an agent that increases Nato3 expression and / or activity. The invention further provides a vector comprising an expression construct according to the invention, optionally a viral vector. The invention additionally provides a cell comprising an expression construct or vector according to the invention. The invention also provides a dopaminergic neuron or a dopaminergic progenitor comprising an expression construct comprising a promoter operably linked to a nucleic acid sequence encoding an agent that increases Nato3 expression and / or activity or a vector comprising said expression construct. The invention further provides a method of screening for a compound that improves dopaminergic function, comprising contacting a dopaminergic neuron according to the invention with a compound, and monitoring said neuron for improvement of one or more parameters of dopaminergic function. Description of the Figures Figure 1. Assessment of hiPSC pluripotency, differentiation into mDA neurons and lentiviral infection. (A) Representative immunocytochemistry images stained for pluripotency markers. Top row from left to right: SSEA4 (magenta), NANOG (green), DAPI (blue), merge. Bottom row from left to right, TRA-1-81 (magenta), OCT4 (green), DAPI (blue). (B) Percentage of cells expressing selected pluripotency markers. (C) Representative image of differentiated hiPSCs into DA neurons labeled with (from left to right) pan-neuronal marker TUJ1 (magenta), TH (green), and DAPI (blue). Scale bar, 20µm. Similar results were observed across at least three independent experiments. (D) Percentage of DA neurons labelled with TH in control and PD differentiated hiPSC lines at DIV35. rounds, n > 500 cells. Graphs show mean ± s.d. Figure 2. NATO3 gene delivery in hiPSC-derived DA neurons. Percentage of neurons co-expressing GFP and TH following the lentiviral-mediated delivery of NATO3 overexpression (lenti N3) or the control construct (lenti GFP). (B) Relative NATO3 expression in PD and control cell lines transduced with the lentivirus expressing NATO3 (NATO3) and the control virus (GFP). Values relative to housekeeping genes and normalized to control cells transfected with the control virus (GFP) determined by RT-qPCR. N=3 independent experiments. Graphs show mean ± s.d. Figure 3. NATO3 overexpression reduces α-Syn levels and aggregates in hiPSC- derived DA neurons. Quantification of (A) α-Syn and (B) Amytracker fluorescence intensity within DA neurons at DIV40 and DIV90. Data compares PD and control cell lines, transfected with NATO3 overexpressing virus (NATO3) and the control virus (GFP). All data from N= 3 independent differentiation experiments. Graphs show mean ± s.d. **p<0.01, ***p<0.001, and ****p<0.0001 (ANOVA with Tukey’s multiple comparison test). ns, not significant. Figure 4. NATO3 overexpression restores aberrant neurite length and complexity in PD DA neurons. (A) Representative images of hiPSC-derived mDA neurons at DIV90 immunostained for TH. Scale bar, 50µm. (B) Neurite length analysis at DIV40 and DIV90. (B) Sholl analysis at DIV40 and DIV90. Data compares PD and control cell lines, transduced with NATO3-expressing lentivirus (NATO3) and the control virus (GFP). All data from N= 3 independent differentiation experiments. Graphs show mean ± s.d. *p<0.05, **p<0.01, and ***p<0.001(ANOVA with Tukey’s multiple comparison test). ns, not significant. Figure 5. Overexpression of NATO3 restores mitochondrial morphology in PD DA neurons. Quantification of mitochondrial volume (A) and sphericity (B) marked by TOM20 at DIV40 and DIV90 in the DN neurons from the control and PD cell lines, expressing NATO3 (NATO3) or the control construct (GFP). All data are from N= 3 independent differentiation experiments. Graphs show mean ± s.d. **p<0.01, ***p<0.001, and ****p<0.0001 (ANOVA with Tukey’s multiple comparison test). ns, not significant. Figure 6. NATO3 overexpression improves the autophagic flux in PD DA neurons. Quantification of P62 (A) and LC3-II (B) fluorescence intensity within hiPSC-derived DA neurons at DIV40 and DIV90. Data compares PD and control cell lines, expressing NATO3 (NATO3) or the control construct (GFP). All data from N= 3 independent differentiation experiments. Graphs show mean ± s.d. **p<0.01, ***p<0.001, and ****p<0.0001 (ANOVA with Tukey’s multiple comparison test). ns, not significant. Figure 7. Stereotactic injection of AAV-Nato3 into the substantia nigra prevents MPTP-induced loss of substantia nigra DA neurons. Wild-type mice received a stereotactic injection of AAV-Nato3 or PBS. One week later, the mice were administered MPTP (20 mg / Kg) or saline (NaCl) IP injections once a day for 5 consecutive days. Mice were sacrificed one month after the first MPTP injection and the DA neurons in the substantia nigra were assessed using anti-TH immunostaining. The number of DA neurons in the injected hemisphere is shown. AAV-Nato3 stereotactic injection prevented MPTP- induced DA neuron loss. The center lines of the box plots indicate the median, the box boundaries represent the 25th and 75th percentiles, and the whiskers represent the minima and maxima. Each dot represents the value of one animal. ***p<0.001 and ***p<0.0001 (two-tailed Mann-Whitney test). ns, not significant. Figure 8. Stereotactic injection of AAV-Nato3 into the substantia nigra prevents the locomotor deficits in an AAV-^^-synuclein model of PD. Wild-type mice received a stereotactic injection of AAV-^^Syn along with either AAV-Nato3 or PBS into one side of the substantia nigra. Stereotactic injections of control AAV with PBS, and AAV-Nato3 with PBS were also performed as controls. Two months post-injection, the locomotor ability of the mice was assessed using a pole test. A. Pole test. The time taken to orient downward (T-turn) and the total time to turn and descend the pole (T-tot) were measured. B. T-turn. Mice injected with AAV-^^Syn displayed significantly impaired locomotor ability, as indicated by an increase in T-turn time. This deficit was reversed by the co- injection of AAV-Nato3. C. T-tot. AAV-^^Syn stereotactic injection significantly increased the T-tot time. Co-injection of AAV-Nato3 showed a tendency to prevent this delay. **p<0.01 and ***p<0.001 (two-tailed Mann-Whitney test). ns, not significant. Figure 9. Stereotactic injection of AAV-Nato3 into the substantia nigra prevents the locomotor deficits in an AAV-^^-synuclein model of PD. Wild-type mice received a stereotactic injection of AAV-^^Syn along with either AAV-Nato3 or PBS into one side of the substantia nigra. Stereotactic injections of control AAV with PBS, and AAV-Nato3 with PBS were also performed as controls. Two months post-injection, the locomotor ability of the mice was assessed using a pole test. A. Pole test. The time taken to orient downward (T-turn) and the total time to turn and descend the pole (T-tot) were measured. B. T-turn. Mice injected with AAV-^^Syn displayed significantly impaired locomotor ability, as indicated by an increase in T-turn time. This deficit was reversed by the co- injection of AAV-Nato3. C. T-tot. AAV-^^Syn stereotactic injection significantly increased the T-tot time. Co-injection of AAV-Nato3 showed a tendency to prevent this delay. **p<0.01 and ***p<0.001 (two-tailed Mann-Whitney test). ns, not significant. Figure 10. Stereotactic injection of AAV-Nato3 into the substantia nigra prevents the loss of DA neurons in the substantia nigra in an AAV-^^-synuclein model of PD. Wild- type mice received a stereotactic injection of AAV-^^Syn along with either AAV-Nato3 or PBS into one side of the substantia nigra. Stereotactic injections of control AAV with PBS, and AAV-Nato3 with PBS were also performed as controls. Two months post-injection, DA neurons in the substantia nigra were examined using anti-TH immunostaining. The number of DA neurons in the injected hemisphere is shown. AAV-Nato3 stereotactic injection prevented the loss of nigral DA neurons induced by AAV-^^Syn. *p<0.05 (two- tailed t-test). ns, not significant. Description of the sequences SEQ ID NO:1 is an exemplary DNA sequence encoding a wild-type human NATO3 transcript SEQ ID NO:2 is an exemplary amino acid sequence for wild-type human NATO3 SEQ ID NOs 3-16 are DNA primer sequences. SEQ ID NO: 17 is a DNA promoter sequence for PGK1. SEQ ID NO: 18 is a DNA sequence for a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Detailed Description General Definitions Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs. In general, the term “comprising” is intended to mean including but not limited to. For example, the phrase “the method comprising administering to the individual an agent” should be interpreted to mean that the method includes administering an agent, but the method may comprise further steps. In some aspects of the disclosure, the word “comprising” is replaced with the phrase “consisting of”. The term “consisting of” is intended to be limiting. For example, the phrase “a method consisting of administering to the individual an agent” should be understood to mean that the method involves only administration of the agent, and no further substances. The singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “an agent” includes two or more instances or versions of such agents. Agent that increases Nato3 activity and / or expression Nato3 is a transcription factor expressed in the mammalian central nervous system and associated with formation of dopaminergic neurons, also known as Ferd3l. Exemplary transcript encoding, coding and amino acid sequences for wildtype human NATO3 are illustrated below with reference to SEQ ID NOs 1 and 2. An agent that increases Nato3 activity and / or expression is any agent that increases Nato3 activity and / or expression directly or indirectly. In particular, the agent may comprise a Nato3 polypeptide or a nucleic acid sequence encoding a Nato3 polypeptide, and thus directly provide for increased Nato3 expression and activity. The agent may cause overexpression of Nato3 polypeptide and / or mRNA. A Nato3 polypeptide includes any known Nato3 polypeptide and any functional variants or fragments thereof. A Nato3 polypeptide may be a mammalian Nato3 polypeptide, such as a human or mouse Nato3 polypeptide, or may be a non-mammalian Nato3 homologue, such as a Drosophila Nato3 homologue, in particular Fer2, depending on the organism of interest. Alternatively, the agent may increase the activity and / or expression of Nato3 directly by interaction with a Nato3 polypeptide or an encoding nucleic acid sequence. The agent may stabilise a Nato3 polypeptide or an encoding mRNA. The agent may be an agonist of Nato3. The agent may increase Nato3 activity by post-translational modification of Nato3, such as phosphorylation, and thus increase Nato3 activity. The agent may provide for increased kinase activity for Nato3. In another embodiment, the agent may inhibit or decrease expression of an antagonist of Nato3. In another alternative, the agent may increase the activity and / or expression of Nato3 indirectly, for example by an effect on an upstream regulator of Nato3 or a downstream target of Nato3, typically an upstream regulator or downstream target in DA neurons. The agent may upregulate expression of Nato3, for example by increasing Nato3 transcription and / or translation. The agent may comprise or encode a transcription factor that upregulates expression of Nato3, such as SREBP1. Alternatively, the agent may increase activity or expression of one or more downstream targets that are typically regulated by Nato3. The agent may comprise or encode a downstream target of Nato3, such as a downstream transcription factor. Examples of downstream targets of Nato3 include Lmx1b, Foxa2, SOD2 and ATP6V0C. The agent may increase Nato3 activity by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400% or greater. The agent may increase expression of Nato3 by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400% or greater. The increase in expression of Nato3 may be present at the mRNA and / or protein level. Typically, the increase is with respect to a suitable control or reference which has not been exposed to the agent. The agent may provide increased Nato3 expression and / or activity compared to that present in a dopaminergic neuron derived from an individual having a neurological disorder discussed herein, such as Parkinson’s disease. The agent may increase or restore Nato3 expression and / or activity to a level observed in a dopaminergic neuron derived from a healthy individual. Nato3 activity may be measured by any suitable assay, for example based on increased SOD2 levels and / or enhanced autophagic flux. Enhanced autophagic flux may be measured for example by decreased LC3 levels. Illustrative assays for Nato3 activity are provided in the Examples. Nato3 expression may be measured at the mRNA and / or protein level. The agent may prevent or reduce one or more phenotypes associated with a neurological disease, as discussed below. The agent may be a small molecule (such as an organic compound with a molecular weight ≤ 1000 Da), a peptide, a protein, an antibody, a polynucleotide, an oligonucleotide, an expression construct, an antisense RNA, an siRNA, an shRNA, an mRNA, an miRNA or a vector, such as a viral vector. The agent may comprise a moiety which specifically targets dopaminergic neurons. Nucleic acids In preferred aspects, the agent comprises a nucleic acid sequence encoding a Nato3 polypeptide. The agent may thus provide for gene therapy with Nato3. One or more control sequences, typically including a promoter, may be operably linked to the encoding nucleic acid sequence to provide an expression construct. One or more other regulatory elements may also be present as well as the promoter. For example, a promoter can be used in tandem with one or more further promoters or enhancers or locus control regions (LCRs). A preferred enhancer is a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), for example the WPRE of SEQ ID NO:18 or a variant thereof. An expression construct allows expression of Nato3. The term “operably linked” refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. The control sequence(s), typically the promoter, may be constitutively or conditionally active. The promoter may be a phosphoglycerate kinase 1 (PGK1) promoter, for example the promoter of SEQ ID NO: 17 or a variant thereof. The promoter may provide for substantially specific expression in dopaminergic neurons. Thus, the promoter may express Nato3 in dopaminergic neurons and not significantly express Nato3 in other cell types. The promoter may thus be a dopaminergic neuron-specific promoter. Examples of dopaminergic neuron-specific promoters include the promoters of the dopamine transporter (DAT), tyrosine hydroxylase (TH) and DOPA decarboxylase (DDC) genes. A nucleic acid sequence encoding Nato3, for example comprised in an expression construct, may be administered via any suitable vehicle. Thus, the nucleic acid sequence may be present in a vector (typically a gene therapy vector), liposome, nanoparticle (for example, a polymeric nanoparticle, solid lipid nanoparticle, or compacted DNA nanoparticle), a dendrimer, polyplex, or polymeric micelles. A vector may be of any type, typically a vector suitable for gene therapy. The vector may be a plasmid vector, a minicircle DNA vector or a viral vector. A viral vector may be comprised in a viral particle. A viral vector may be based on any suitable virus, such as herpes simplex virus, adenovirus, adeno-associated virus or lentivirus. The virus may be of any serotype or serogroup or pseudotype. A preferred lentiviral vector pseudotype is VSV-G. An adeno-associated viral vector may be of the serotype AAV6. An AAV6 vector may further comprise a PGK-1 promoter and / or a WPRE enhancer as discussed above. A viral vector may comprise two inverted terminal repeats (ITRs), preferably one at each end of the genome. An ITR sequence acts in cis to provide a functional origin of replication and allows for the integration and excision of the vector from the genome of a cell. A vector may be prepared by standard means known in the art for the provision of vectors for gene therapy. Thus, well established public domain transfection, packaging and purification methods can be used to prepare a suitable vector preparation. The invention also relates to an expression construct comprising a promoter operably linked to a nucleic acid sequence encoding an agent that increases Nato3 expression and / or activity as a product per se. The invention additionally relates to a vector comprising such an expression construct, preferably a viral vector. The viral vector may be based on any virus discussed above, such as an adeno-associated virus or lentivirus. The above discussion relating to expression constructs and vectors for gene therapy is also applicable to provision of nucleic acids encoding upstream regulators or downstream targets of Nato3. Neurological disorder A neurological disorder that may be prevented or treated according to the invention typically comprises impaired function of dopaminergic neurons, also described as impaired dopaminergic function. The existence of an impaired dopaminergic function may be known or inferred from symptoms or examination of an individual. For example, the existence of Parkinsonian motor disability may allow for diagnosis of impaired dopaminergic function, in particular in Parkinson’s disease. Brain imaging may be used to detect changes in the substantia nigra indicative of impaired dopaminergic function, for example using methods such as PET and MRI. The agent that increases activity and / or expression may increase dopaminergic function or restore normal dopaminergic function, to prevent or treat the disorder. The agent may reduce abnormalities observed in brain imaging or restore a normal brain imaging pattern, for example in the substantia nigra. The neurological disorder may be a neurodegenerative disorder or a neurodevelopmental disorder. The neurological disorder may be a cognitive disorder, such as a dementia. The neurological disorder may be associated with epilepsy. The neurological disorder may comprise overexpression and / or aggregation of α- synuclein, including in dopaminergic neurons. The aggregates may be fibrillar in form. The neurological disorder may comprise the presence of Lewy bodies and / or Lewy neurites, including in dopaminergic neurons. The overexpression and / or aggregation of α- synuclein may be the result of one or more mutations in the α-synuclein gene. The neurological disorder may comprise reduced expression or inactivation of Nato3 or of any upstream regulator or downstream target of Nato3, including those discussed above. The reduced expression or inactivation may be the result of one or more mutations in the relevant gene. The neurological disorder may comprise V-ATPase dysfunction, which is characteristic of many neurodegenerative diseases, as described for example in Higashida et al (2017) and Song et al. (2020). The V-ATPase may be ATP6V0C. The disorder may be a neurodevelopmental disorder associated with mutation of the V-ATPase ATP6V0C, a downstream target of Nato3, see for example Mattison et al (2022). The agent may prevent or reduce one or more of the following phenotypes in the neurological disease: neurodegeneration of dopaminergic neurons in the substantia nigra, synapse degeneration, mitochondrial dysfunction, defective autophagic flux, oxidative stress, α-synuclein overexpression and / or aggregation and / or presence of Lewy bodies. The above phenotypes are typically ameliorated in dopaminergic neurons, preferably dopaminergic neurons of the substantia nigra. The agent may prevent or reduce loss of dopaminergic neurons of the substantia nigra. The agent may prevent or reduce locomotor deficits. The agent may prevent or reduce motor dysfunction. The agent may prevent or reduce loss of dopaminergic neurons of the substantia nigra and prevent or reduce locomotor deficits or motor dysfunction. The agent may decrease α-synuclein levels. The agent may increase efficiency of autophagic flux, which may decrease α-synuclein levels. The agent may reduce levels of autophagy markers, such as LC3 and / or P62. The agent may increase mitochondrial volume and / or decrease mitochondrial sphericity. The agent may reduce mitochondrial dysmorphia. The agent may restore neurite length and / or complexity. Exemplary methods for evaluation of the above phenotypes are illustrated in the examples. The effect of the agent on the above phenotypes may be evaluated in dopaminergic neurons differentiated from iPS cells generated from somatic cells of an individual having the neurological disease. Examples of neurodegenerative disorders include Amytrophic lateral sclerosis (ALS), Huntington’s disease, Multiple sclerosis, Alzheimer’s disease, Lewy body dementia, and Parkinson’s disease. Alzheimer’s disease may comprise Lewy body pathology. A particularly preferred disorder is Parkinson’s disease. Parkinson’s disease may be pre-clinical, prodromal or clinical. Symptoms of prodromal Parkinson’s disease may include REM sleep behavior disorder (RBD). Clinical symptoms of Parkinson’s disease are well described, and include Parkinsonian motor symptoms (Kalia & Lang, 2015; Noyce et al., 2016). Parkinson’s disease may be characterized by one or more of the following phenotypes: selective neurodegeneration of dopaminergic neurons in the substantia nigra, synapse degeneration, mitochondrial dysfunction, defective autophagic flux, lysosomal disruption, oxidative stress, α-synuclein overexpression and / or aggregation, and presence of Lewy bodies. Parkinson’s disease may be familial or sporadic. Parkinson’s disease may be associated with one or more mutations providing a predisposition to the disease, for example one or more mutations in α-synuclein, typically mutations leading to overexpression and / or aggregation of α-synuclein. An example of a highly penetrant α-synuclein mutation in Parkinson’s disease is A53T, as reported in Oliveira et al., 2021. Individual The individual may be any individual that has a neurological disorder. The neurological disorder may be selected from any discussed above, preferably Parkinson’s disease. The individual may show signs or symptoms of the neurological disorder. In this case, administration of the agent may treat the disorder. That is, administration of the agent may reduce or abolish the clinical signs or symptoms of the disorder, or stop them from progressing. Signs and symptoms of Parkinson’s disease include those discussed above and below and any others known in the art, as described for example in (Kalia & Lang, 2015). Early signs may include one or more of constipation, RBD, EDS, hyposmia, and depression. Clinical symptoms may include motor symptoms such as bradykinesia, rigidity and / or tremor. Advanced motor symptoms may include one or more of dysphasia, postural instability, freezing of gait, falls. Clinical non-motor symptoms may include one or more of pain, fatigue, urinary symptoms, dementia, psychosis. The agent may prevent or reduce loss of dopaminergic neurons of the substantia nigra in Parkinson’s disease. The agent may prevent or reduce locomotor deficits or motor dysfunction in Parkinson’s disease. The agent may prevent or reduce loss of dopaminergic neurons of the substantia nigra and prevent or reduce locomotor deficits or motor dysfunction in Parkinson’s disease. The individual may be an individual that is at risk of developing the disorder, for example based on familial predisposition. The individual may have a mutation in a gene associated with the disorder, but not yet show clinical signs or symptoms of a disorder. In other words, the individual may be presymptomatic. In this case, administration of the agent may prevent the development of clinical signs or symptoms of the disorder. The individual may exhibit impaired dopaminergic function, in particular impaired dopaminergic function in the substantia nigra. The individual may have about 90% or less, about 80% or less, about 70% or less, about 60% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, or about 10% or less dopaminergic function relative to a reference value, such as a healthy individual.The individual may exhibit one or more of the following phenotypes: neurodegeneration of dopaminergic neurons in the substantia nigra, synapse degeneration, mitochondrial dysfunction, defective autophagic flux, oxidative stress, α-synuclein overexpression and / or aggregation and / or presence of Lewy bodies. The individual is typically a mammal. Preferably, the mammal is a human. The individual may be of any age. For example, the individual may be a juvenile. The individual may, for example, be an adult. The individual may, for example, be a prenatal individual. In other words, the individual may be in utero. That is, the individual may be a foetus. The prenatal individual may, for example, be presymptomatic. In other words, the prenatal individual may have a mutation that is linked to a neurological disorder, but not yet show signs of said neurological disorder. In one aspect of the present invention, the individual has been diagnosed with a neurological disorder, such as any neurological disorder described above, preferably Parkinson’s disease. Administration The agent may be administered by any route. Suitable routes include, but are not limited to, the intravenous, intrathecal, intracerebral ventricular, intramuscular, intraperitoneal, subcutaneous, intradermal, transdermal, nasal and oral / buccal routes. When the individual is a prenatal individual, the agent may be administered to the individual in utero. In other words, the agent may be administered directly to the individual while the individual is in their mother’s uterus. The agent may be one that is capable of crossing the placenta. In this case, the agent may be administered to the individual by administering it to the mother of the individual. The agent may be comprised in a composition that comprises a physiologically acceptable carrier or diluent. Typically, such compositions are prepared as liquid suspensions of the agent. The agent may be mixed with an excipient which is pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, of the like and combinations thereof. In addition, if desired, the pharmaceutical compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, and / or pH buffering agents. The agent is administered in a manner compatible with the dosage formulation and in such amount will be therapeutically effective. The quantity to be administered depends, for example, on the subject to be treated, the nature of the neurological disorder (e.g. symptomatic or presymptomatic), and so on. Precise amounts of the agent required to be administered may depend on the judgement of the practitioner and may be peculiar to each subject. Cells The invention further relates to a cell comprising an expression construct or vector of the invention, and its use in preventing or treating a neurological disorder. The cell may be a dopaminergic neuron or a dopaminergic progenitor. Cell-based strategies for treatment of PD are described for example in Wang et al., 2023. A dopaminergic progenitor may be described herein as a neural stem cell able to generate a dopaminergic neuron. A dopaminergic neuron may also be described herein as a finally differentiated post-mitotic cell which is electrically excitable and is capable of dopamine biosynthesis. A dopaminergic neuron may have one or more features specific to dopaminergic neurons of the substantia nigra. A dopaminergic neuron may have an ability to synthesise and release dopamine, to exhibit axonal projections and arborization, to make projections to the striatum, and / or to make synaptic connections with neurons that express D1-family dopamine receptors and D2-family dopamine receptors. The cell may be a mammalian or a human cell. The cell may be derived from a cell of an individual as discussed above that has a neurological disorder, or is at risk of developing such a disorder, preferably wherein the disorder is Parkinson’s disease. The cell may comprise one or more mutations associated with the neurological disorder as discussed above, for example one or more mutations in α-synuclein, such as A53T. The cell may be derived from an induced pluripotent stem cell (iPS cell) generated from a somatic cell of the individual, such as a fibroblast (typically a skin fibroblast), or a blood cell. The iPS cell may then be differentiated into a dopaminergic neuron or a dopaminergic progenitor cell (The skilled person is aware of suitable methods to generate iPS cells from somatic cells and differentiate iPS cells into dopaminergic neurons or dopaminergic progenitors, as described for example in Tofoli et al., 2019, and Yamanaka, 2020. The differentiated cell may then be modified to express an agent, that increases Nato3 expression and / or activity according to the invention, such as by introduction of a nucleic acid sequence encoding a Nato3 polypeptide or an expression construct or vector of the invention, to provide a cell according to the invention. In this way, the cell may comprise increased Nato3 expression and / or activity, for example compared to a dopaminergic neuron of an individual having a neurological disorder described herein. The cell may comprise increased Nato3 expression and / or activity compared to a dopaminergic neuron of an individual from which it was derived. The cell may comprise Nato3 expression and / or activity restored to a level as present in a dopaminergic neuron of a healthy individual, or higher. A cell of the invention typically comprises a nucleic acid sequence encoding a Nato3 polypeptide at a distinct chromosomal location to the endogenous Nato3 gene. The cell may comprise an integration of a viral vector comprising a nucleic acid sequence encoding a Nato3 polypeptide, such as an adeno-associated virus vector or lentiviral vector. The invention further provides a plurality of cells according to the invention in which at least 30%, 40%, 50%, 60%, 70%, more preferably at least 80%, particularly preferably at least 90% of the cells comprise increased expression and / or activity of Nato3, for example compared to a dopaminergic neuron of an individual having a neurological disorder described herein. At least 30%, 40%, 50%, 60%, 70%, more preferably at least 80%, particularly preferably at least 90% of the cells may comprise Nato3 expression and / or activity restored to a level as present in a dopaminergic neuron of a healthy individual, or higher. The cell (typically a plurality of cells) may be used to prevent or treat a neurological disorder, by their administration to an individual having a neurological disorder described herein, or at risk of such a disorder. The administration may be by transplantation into the brain of the individual. Thus, a dopaminergic neuron or dopaminergic progenitor of the invention, for example generated as discussed above, may be transplanted into the brain of an individual at risk of, or having the neurological disorder. The transplantation may be into the striatum. The skilled person is aware of suitable methods for transplantation of dopaminergic neurons and progenitors into the brain, as described for example in (Parmar et al., 2020). The invention also relates to a method of producing a dopaminergic neuron or a dopaminergic progenitor cell, comprising differentiating an iPS cell into a dopaminergic neuron or a dopaminergic progenitor cell, and introducing a nucleic acid sequence encoding a Nato3 polypeptide or an expression construct or vector of the invention into the differentiated cell. The invention further relates to a dopaminergic neuron or a dopaminergic progenitor cell obtainable by the above method. The invention further relates to a non-human organism, typically a mammal, such as a rodent (for example a mouse or rat), which comprises in its genome an expression construct comprising a promoter operably linked to a nucleic acid sequence encoding an agent that increases Nato3 expression and / or activity or a vector comprising said expression construct. Preferably, the nucleotide sequence encodes a Nato3 polypeptide. The non-human organism may serve as a model for effects of increasing Nato3 expression and / or activity. The non-human organism may further exhibit or model one or more features of a neurological disorder, such as any disorder described here, for example Parkinson’s disease. The non-human organism may comprise one or more mutations in a gene associated with the disorder, typically a homologue of a human gene associated with the disorder, and corresponding mutation(s) to mutation(s) found in the human gene in the disorder, for example any mutation discussed above, such as A53T in α-synuclein. The invention additionally relates to a method of screening for a compound that improves dopaminergic function, comprising contacting a dopaminergic neuron or non- human organism of the invention with a compound, and monitoring said neuron for improvement of one or more parameters of dopaminergic function. Typically a plurality of neurons are contacted with the compound and monitored. The method may comprise identifying a compound that improves dopaminergic function. The compound may improve dopaminergic function by further increasing Nato3 expression and / or activity. The method may comprise monitoring any suitable one or more parameter(s) of dopaminergic function. The one or more parameters may be selected from neurite length and / or complexity, mitochondrial function, autophagic flux, and α-synuclein expression and / or aggregation. Embodiments of the invention 1. An agent that increases Nato3 expression and / or activity for use in a method for preventing or treating a neurological disorder. 2. The agent according to embodiment 1, which comprises an expression construct comprising a promoter operably linked to a nucleic acid sequence encoding an agent that increases Nato3 expression and / or activity, or a vector comprising said expression construct. 3. The agent according to embodiment 2, which is a viral vector, optionally an adeno associated virus (AAV) vector or a lentiviral vector. 4. The agent according to embodiment 3, which is a lentiviral vector. 5. The agent according to any one of the preceding embodiments, which is a cell comprising an agent that increases Nato3 expression and / or activity, an expression construct comprising a promoter operably linked to a nucleic acid sequence encoding an agent that increases Nato3 expression and / or activity, or a vector comprising said expression construct. 6. The agent according to any one of the preceding embodiments, which comprises a Nato3 polypeptide or a nucleic acid sequence encoding a Nato3 polypeptide. 7. The agent according to embodiment 5 or 6, which is a dopaminergic neuron or a dopaminergic progenitor, optionally derived from a patient with a neurological disorder. 8. The agent for use according to any one of the preceding embodiments, wherein the neurological disorder is a neurodegenerative disorder, optionally characterised by overexpression and / or aggregation of α-synuclein and / or the presence of Lewy bodies. 9. The agent for use according to embodiment 8, wherein the neurodegenerative disorder is Parkinson’s or Alzheimer’s disease. 10. The agent for use according to any one of the preceding embodiments, wherein administration of the agent prevents or reduces one or more of the following phenotypes associated with the neurological disease: neurodegeneration of dopaminergic neurons in the substantia nigra, synapse degeneration, mitochondrial dysfunction, defective autophagic flux, oxidative stress, α- synuclein overexpression and / or aggregation and / or Lewy bodies. 11. The agent for use according to any one of the preceding embodiments, which is administered to a patient comprising an overexpression and / or aggregation of α- synuclein and / or Lewy bodies, optionally in dopaminergic neurons. 12. The agent for use according to any of the preceding embodiments, wherein the patient comprises an α-synuclein mutation causing overexpression and / or aggregation of α-synuclein, optionally A53T. 13. A method of preventing or treating a neurological disorder or increasing dopaminergic function comprising administering an agent that increases Nato3 expression and / or activity to a patient; optionally wherein the agent, administration, neurological disorder or patient are as defined in any one of the preceding embodiments. 14. Use of an agent that increases Nato3 expression and / or activity in the manufacture of a medicament for preventing or treating a neurological disorder; optionally wherein the agent, administration, neurological disorder or patient are as defined in any one of embodiments 1-12. 15. An expression construct comprising a promoter operably linked to a nucleic acid sequence encoding an agent that increases Nato3 expression and / or activity. 16. An expression construct according to embodiment 15, wherein said nucleic acid sequence encodes a Nato3 polypeptide 17. A vector comprising an expression construct according to embodiment 15 or 16, optionally a viral vector, optionally wherein the viral vector is an adeno associated virus (AAV) vector or a lentiviral vector. 18. The viral vector according to embodiment 15, which is a lentiviral vector. 19. A cell comprising an expression construct or vector according to any one of embodiments 15 to 18. 20. A dopaminergic neuron or a dopaminergic progenitor comprising an expression construct comprising a promoter operably linked to a nucleic acid sequence encoding an agent that increases Nato3 expression and / or activity or a vector comprising said expression construct. 21. The dopaminergic neuron or a dopaminergic progenitor according to embodiment 20, wherein said vector is a viral vector, optionally an adeno associated virus (AAV) vector or a lentiviral vector. 22. The dopaminergic neuron or a dopaminergic progenitor according to embodiment 20 or 21, wherein said nucleic acid sequence encodes a Nato3 polypeptide. 23. A non-human organism comprising in its genome an expression construct comprising a promoter operably linked to a nucleic acid sequence encoding an agent that increases Nato3 expression and / or activity or a vector comprising said expression construct. 24. The non-human organism of embodiment 23, which is a rodent, optionally a mouse or rat. 25. The non-human organism of embodiment 23 or 24, wherein said nucleic acid sequence encodes a Nato3 polypeptide. 26. A method of screening for a compound that improves dopaminergic function, comprising contacting a dopaminergic neuron as defined in any one of embodiments 20 to 22 or a non-human organism as defined in any one of embodiments 23-25 with a compound, and monitoring said neuron for improvement of one or more parameters of dopaminergic function. 27. The method of embodiment 26, wherein the one or more parameters of dopaminergic function are selected from neurite length and / or complexity, mitochondrial function, autophagic flux, and α-synuclein expression and / or aggregation. 28. The method of embodiment 26 or 27, wherein the dopaminergic neuron is derived from a patient at risk of, or having a neurological disorder, optionally Parkinson’s disease. Examples Materials and Methods Plasmids and cloning The lentiviral vector for NATO3 overexpression contained the PGK promoter and the NATO3 (NM_152898.2) coding sequence tagged at the N-terminus with an HA epitope tag, followed by an internal ribosome entry site sequence (IRES), the EGFP coding sequence, and the woodchuck posttranscriptional regulatory element (WPRE). The synthetic construct (Plasmid #1) was custom-made for us by Eurofins and cloned into a lentiviral transfer plasmid backbone, kindly provided by Dr. Bernard Schneider (Plasmid #2). The plasmid backbone was digested at KflI and XhoI restriction enzyme sites and the NATO3 construct was cloned using Gibson assembly (E5510S, NEB) as per manufacturer instructions (Plasmid #3). This final construct was referred to as NATO3 lentivirus. The lentiviral transfer plasmid backbone contained GFP under the PGK promoter and was used as a control (Plasmid #2). This control plasmid was referred to as GFP lentivirus. The lentivirus production was done at the EPFL Bertarelli Foundation Gene Therapy Platform. hiPSC culture and differentiation into midbrain dopaminergic neurons hiPSC lines were obtained from the NINDS Repository at the Coriell Institute for Medical Research (PD patient harboring the SNCA A53T point mutation, cat. no. ND50086; mutation corrected isogenic line, cat. no. ND50085). hiPSCs were maintained on Geltrex (A1413302, Thermo Fisher Scientific) in StemFlex basal medium supplemented with 10% StemFlex supplement x10 (A3349401, Thermo Fisher Scientific) and passaged using the Versene solution (BE17‐711E, Lonza). Differentiation of hiPSCs into midbrain dopaminergic neurons was performed as previously described(Tofoli et al., 2019). In summary, hiPSCs were seeded onto Geltrex- coated six-well plates, expanded until >80% confluency, and StemFlex media was changed on day 0 to in vitro (DIV) neuronal induction media consisting of knockout DMEM with knockout serum replacement supplemented with LDN193189 (100 nM) and SB431542 (10 uM). From DIV1, Sonic Hedgehog (SHH) C25II (100 ng / ml), Purmorphamine (2 uM), and Fibroblast growth factor 8 (FGF8; 100 ng / ml) were added, followed by the addition of CHIR99021 (3 uM) from DIV3. Starting at DIV5, the KSR medium, consisting of DMEM KO (10829-018, Thermo Fisher Scientific) with 1% MEM NEAA 100x (11140035, Thermo Fisher Scientific), 1% Glutamax 100x (35050038, Thermo Fisher Scientific), 0.1% ^-Mercaptoethanol 1000x (21985-023, Thermo Fisher Scientific) and 15% KSR (10828028, Thermo Fisher Scientific) as incrementally shifted to N2 medium, consisting of DMEM F12 (11320033, Thermo Fisher Scientific) with 1% Glutamax 100x (35050038, Thermo Fisher Scientific) and N2 supplement 100x (17502048, Thermo Fisher Scientific), (25%, 50%, 75%) every 2 days, and SB431542 supplementation was discontinued. From DIV7 the media were only supplemented with LDN (100 nM) and CHIR99021 (3 uM). On DIV11 and onwards, the medium was changed to neuronal maturation medium consisting of Neurobasal (21103049, Thermo Fisher Scientific) with 2% B27 supplement 50x (17504044, Thermo Fisher Scientific) and 1% Glutamax (35050038, Thermo Fisher Scientific), supplemented with Brain Derived Neurotrophic Factor (BDNF; 20 ng / ml), ascorbic acid (200 uM), Glial Derived Neurotrophic Factor (GDNF; 20 ng / ml), transforming growth factor beta 3 (TGFb3; 1 ng / ml), dibutyryl cAMP (db cAMP; 250 uM) and DAPT (10 uM). Additionally, CHIR99021 (3 uM) was supplemented on DIV11 and DIV12. On DIV20, the cells were dissociated using accutase (561527, BD Biosciences) and seeded at a density of 2x105cells / cm2 onto 24-well plates coated with Geltrex. Y27632 (10uM) was added to the medium after passages. All mediums contained 1% penicillin-streptomycin and the cells were cultured in a humidified atmosphere of 5% CO2 and 95% air at 37°C. Lentiviral infection was performed on dopaminergic neurons derived from hiPSCs on DIV30. Infection was performed at a Multiplicity of Infection (MOI) of 5. The day after transfection, the medium was changed and neurons were cultured neuronal maturation medium until the experimental time points. RT- qPCR Total RNA was extracted from cells using the TRIzol Reagent (Life Technologies) following the manufacturer’s instructions and was treated with Turbo DNase-free kit (AM1907, Thermo Fisher Scientific). DNA removal and reverse transcription was performed using the Maxima H Minus cDNA Synthesis Master Mix with dsDNase (M1682, Thermo Fisher Scientific) as per the manufacturer’s protocol. Quantitative PCR analyses were performed in a QuantStudio 5 PCR machine using the PowerTrack SYBR Green Master Mix (C14512, Applied Biosystems). All sample analyses were carried out in triplicate. Relative expression levels were normalized to the expression of housekeeping genes. Primers were designed with the Primer-BLAST online tool, and the primer sequence information is presented in Table 1. Immunohistochemistry Mice were anesthetized and perfused with 0.2% Heparin in PBS at RT followed by ice- cold 10% formalin solution (Sigma HT501128-4L). The brains were dissected and post- fixed for 72h in fresh 4% paraformaldehyde (PFA) (Thermo Fisher Scientific #28906) before being embedded in low melting point agarose (Thermo Fisher Scientific #R0801) and sliced into 50 µm thick sections using a sliding vibratome. Immunostaining was performed on floating sections. For cell samples, cells were cultured on Geltrex-coated coverslips and fixed with cold 4% PFA (Thermo Fisher Scientific #28906) for 15 minutes at RT and washed three times for 10 minutes with PBS. Mice and cell samples were incubated in a permeabilization-blocking buffer (2% NGS, 0.1% Triton X100 in PBS) for 30 minutes at RT. The samples were then incubated with primary antibodies in an antibody solution (2% NGS, 0.1 % Tween 20 in PBS) overnight at 4ºC. Following the primary antibody incubation, the samples were washed with 0.05% Tween-PBS three times for 30 minutes. Then, the samples were incubated with the corresponding secondary antibodies overnight at 4ºC for mice brain sections, and for 2 hours at room temperature for cell samples. After three washes of 30 minutes with 0.05% Tween-PBS and a final wash of 30 minutes with PBS, the samples were mounted with Vectashield Plus (Vectorlabs, Cat. # H-1900) for mice sections and Vectashield Plus with DAPI (Vectorlabs, Cat. # H-2000) for cell samples. Amytracker stained cells were incubated for 30 minutes with Amytracker 630 (Ebba Biotech) followed by 3 washes of 30 minutes each with PBS, prior to mounting. Table 2 provides a detailed description of antibodies used in the study. Microscopy and image analysis Images of mouse brain slices were captured using a Nikon Ti / CSU-W1 Spinning Disc Confocal microscope with a 63x air objective. Cellular images were captured with a Leica DM5000 fluorescence microscope at 20x magnification. All image analyses were conducted using ImageJ software (v.1.54f). Fluorescence intensity was measured within the regions of interest (ROI) based on the neuronal soma of DA neurons identified by TH positive signal and co-localizing with GFP signal in the case of transfected neurons. Neurite length was quantified using the NeuroAnatomy plugin for ImageJ. Mitochondria, labeled with TOM20 antibody in hiPSCs-derived dopaminergic neurons, were imaged using a Nikon Ti / CSU-W1 Spinning Disc Confocal microscope with a 100x oil- immersion objective and analyzed using the Mitochondria Analyzer plugin for ImageJ. Statistical analysis Statistical comparisons were performed using GraphPad Prism 6 software (Graph Pad Software Inc., CA). Since most data showed non-normal distribution, Mean differences were analyzed using two-tailed Mann–Whitney test for two group comparison and ANOVA followed by Tukey’s multiple comparison test was used to compare among groups. Differences were considered significant at *p < 0.05, **p < 0.01, ***p < 0.001, ***  < 0.0001 while “ns” indicates not significant. Pairwise comparisons are shown as brackets. Table 1. Primer sequences employed for qPCR Gene name Forward sequence Reverse sequence Atp6v0c ATCCTCTCCACAAAGTAGT GCACTAGGACACTGCAC CCTT (SEQ ID NO:3) ATT (SEQ ID NO:4) Actin CTAAGGCCAACCGTGAAAA CACAGCCTGGATGGCTA GAT (SEQ ID NO:5) CGT (SEQ ID NO:6) Gapdh TCCATGACAACTTTGGCAT CAGTCTTCTGGGTGGCA TG (SEQ ID NO:7) GTGA (SEQ ID NO:8) NATO3 GCGAAAGGAAGCGGATGTT CGGGACAGCCTTTTCTCG C (SEQ ID NO:9) TA (SEQ ID NO: 10) GAPDH AAGAAGGTGGTGAAGCAG GTCAAAGGTGGAGGAGT GC (SEQ ID NO:11) GGG (SEQ ID NO:12) ACTB AAGTGTGACGTGGACATCC CTCAGGCAGGAAAGACA G (SEQ ID NO:13) CCC (SEQ ID NO:14) TBP TGCACAGGAGCCAAGAGTG CACATCACAGCTCCCCA AA (SEQ ID NO:15) CCA (SEQ ID NO:16) Table 2. Primary and secondary antibody information. PRIMARY ANTIBODIES ANTIGEN / E HOST REFERENCE # COMPANY DILUTION PITOPE SPECIES NAME TH Rabbit AB152 Merck 1:1000 Millipore TH Mouse 22941 Immunostar 1:1000 ATP6V0C Rabbit ab230323 Abcam 1:500 HMGA1B Rabbit ab4078 Abcam 1:500 LC3 Mouse 0260-100 / LC3- Nanotools 1:500 2G6 P62 Mouse H00008878-M01 Abnova 1:500 (SQSTM1) UBQ Mouse sc-8017 Santa Cruz 1:500 Biotech TUJ1 Chicken TUJ7947980 AVES 1:500 α- Mouse 610787 BD 1:500 SYNUCLEI Biosciences N Amytracker6 N / A Amytracker630 Ebba biotech 1:1000 30 TOM20 Mouse sc-17764 Santa Cruz 1:500 Biotech NANOG Goat AF1997 R&D 1:500 Systems SSEA Mouse MC-813-70 DSHB 1:40 TRA-1-81 Mouse MAB4381 Merck 1:500 Millipore OCT4 Rabbit 09-0023 Stemgent 1:500 SECONDARY ANTIBODIES FLUOROP HOST REFERENCE # COMPANY DILUTION HORE SPECIES Alexa Fluor Rabbit A-21070 Thermo 1:500 633 Fisher Alexa Fluor Rabbit A-11036 Thermo 1:500 568 Fisher Alexa Fluor Rabbit A-11008 Thermo 1:500 488 Fisher Alexa Fluor Mouse A-21050 Thermo 1:500 633 Fisher Alexa Fluor Mouse A-11004 Thermo 1:500 568 Fisher Alexa Fluor Mouse A-11001 Thermo 1:500 488 Fisher Alexa Fluor Chicken A-11041 Thermo 1:500 568 Fisher Alexa Fluor Goat A32814 Thermo 1:500 488 Fisher Example 1 – Differentiation of hiPSCs into midbrain dopaminergic neurons and overexpression of NATO3 Human induced pluripotent stem cell (hiPSC) technology has become a great ally for studying disorders with monogenic or complex genetic causes, as is the case of PD. hiPSCs provide access to human dopaminergic (DA) neurons from Parkinson’s disease (PD) patients that retain genetic risk variants and offer a tool that circumvents the limited similarity between animal models and humans (Singh Dolt et al., 2017). Our aim in this study was to explore if human NATO3 overexpression has a neuroprotective effect in DA neurons in the context of PD. The A53T mutation in the SNCA gene, encoding α-Synuclein (α-Syn), causes autosomal dominant PD (Polymeropoulos et al., 1997). To investigate the neuroprotective potential of NATO3, a hiPSC line derived from a patient carrying the A53T α-Syn point mutation and its isogenic mutation-corrected control line were obtained from the NINDS Repository at the Coriell Institute for Medical Research. The pluripotent state of these hiPSC lines was confirmed through immunocytochemical analysis, demonstrating the expression of pluripotency markers, including OCT4, TRA-1-81, SSEA4, and NANOG, in approximately 99-94% of both PD and control hiPSC cells (Fig.1A and B). The hiPSC lines were differentiated into midbrain DA (mDA) neurons following the established protocol (Tofoli et al., 2019). The differentiation was initiated at day 0 in vitro (DIV0) and completed by DIV30. The efficacy of the differentiation process was validated through immunostaining against TH (tyrosine hydroxylase), a marker specific to dopaminergic neurons. The protocol resulted in the generation of approximately 20% of cells differentiated into TH-positive neurons (Fig.1C and D). NATO3 overexpression in the PD and control cell lines was achieved with two different lentiviral constructs pseudotyped with VSV-G envelope: one expressing NATO3-IRES- GFP under the PGK promoter (referred to as NATO3) and a control virus expressing GFP under the PGK promoter (referred to as GFP). The GFP plasmid served as a control to account for any potential effects of lentiviral transfection on the cells. Lentiviral infection was performed on DIV30. Over 90% of the DA neurons were infected by the lentivirus, judging from the number of cells co-expressing TH and GFP (Fig.2A). NATO3 endogenous levels and NATO3 overexpression were measured in both cell lines, by RT- qPCR (Fig.2B). NATO3 endogenous levels were similar in both control and PD lines, with a clear increase in the transfected cells. Lentiviral infection gave significantly higher efficiency of transduction compared to AAV infection in initial experiments (data not shown). However, it is likely that AAV transduction can be improved. Example 2 - NATO3 overexpression reduces α-Synuclein levels and protein aggregation in PD neurons We identified that NATO3 overexpression ameliorates various Parkinson’s disease-related characteristics in hiPSC-derived dopaminergic neurons, firstly α-Synuclein levels and protein aggregation. SNCA encodes the pre-synaptic neuronal protein α-Syn, the major component of Lewy bodies (Spillantini et al., 1997; Baba et al., 1998). A53T mutation in α-Syn is associated with autosomal dominant, early-onset PD (Polymeropoulos et al., 1997). The A53T mutation increases α-Syn protein stability and tendency to form protofibrils (Conway et al., 2000). Aligning with these notions, it has previously been shown that α-Syn protein levels were increased in the neurons derived from a hiPSC line carrying the SNCA A53T mutation compared to the control culture (Kouroupi et al., 2017). Immunohistochemistry revealed an increased level of α-Syn in DA neurons derived from our PD hiPSCs compared to those from the control hiPSCs at DIV40 and DIV90, congruent with the previous report (Kouroupi et al., 2017). Whereas the expression of the control construct did not alter this difference, the overexpression of NATO3 reduced α-Syn levels in PD neurons to the levels of the control cells at DIV40 and further below the control levels at DIV90. NATO3 expression temporarily increased α-Syn levels in the control neurons at DIV40, but this trend was reversed at DIV90 (Fig.3A). These results indicate that NATO3 overexpression reduces the accumulation of α-Syn protein in human mDA neurons, effectively preventing aberrant α-Syn overaccumulation caused by the A53T mutation. The presence of protein aggregates, notably Lew bodies and the aggregates of the microtubule-associated protein Tau, is a pathological hallmark of PD (Goedert et al., 2013). We next monitored protein aggregates in hiPSC-derived mDA neurons using the Amytracker 630 marker, which stains repetitive arrangement of β-sheet structures typically found in amyloids (Morten et al., 2022). Amytracker stain revealed a progressive accumulation of aggregates in PD neurons. Whereas no difference was observed between PD and control neurons at DIV40, Amytracker fluorescence levels were significantly increased in PD neurons at DIV90. NATO3 overexpression reversed this increase observed at DIV90, as well as reduced the Amytracker levels in the control cells at both time points (Fig.3B). Taken together, these data indicate that NATO3 overexpression reduces α-Syn levels and prevents the accumulation of misfolded proteins. Example 3 - NATO3 overexpression reverses aberrant neurite morphology in PD neurons Axonal degeneration has a central role in PD pathogenesis. Decreasing the number of DA neuron projections in the striatum leads to the motor symptoms of the disease (Hornykiewicz, 1998; Cheng et al., 2010). Dystrophic neurites have been identified in the brain of A53T patients (Duda et al., 2002). This trait has also been observed in DA neurons derived from the hiPSCs carrying the α-Syn A53T mutation (Kouroupi et al., 2017; Czaniecki et al., 2019). We analyzed the length and complexity of hiPSC-derived DA neurons and observed the pathological changes in neurites in PD cells as reported. At both DIV40 and 90, the length of the neurites was reduced in PD neurons compared to the control neurons, and the Sholl analysis revealed reduced neurite complexity in PD neurons than the control (Fig.4A and B) The overexpression of NATO3 extended the neurites and increased their complexity in the PD cells to the levels of the control cells at both time points (Fig.4A and C). These results reveal that NATO3 overexpression counteracts the detrimental effect of the α-Syn A53T mutation in the development and maintenance of neurites. Neurite length is closely related to neuronal function, as longer neurites have an increased capacity for synaptic connections and the transmission of signals. Therefore, NATO3 overexpression likely restores neuronal functions that are impaired by the α-Syn A53T mutation. Example 4 - NATO3 overexpression reverses dysmorphic features of mitochondria in PD neurons Mitochondrial morphology, behavior, and functions are disrupted in cells with PD (Henrich et al., 2023). Abnormal changes in mitochondrial shape have been linked to α- Syn oligomers and aggregates (Xie & Chung, 2012; Plotegher et al., 2014), indicating a mechanistic link between α-Syn and mitochondrial morphology in PD. We assessed mitochondrial morphology in hiPSC-derived DA neurons by immunostaining for TOM20, the marker of the mitochondrial outer membrane. Mitochondrial volume was decreased, and sphericity was increased in the PD neurons compared to the control cells (Fig.5A and B), suggesting enhanced mitochondrial fission in the PD line. Fission is required for the removal of damaged organelles by autophagy (Twig & Shirihai, 2011; Pickrell & Youle, 2015), thus, this difference in mitochondrial morphology could be due to an accumulation of damaged mitochondria as a result of a failure in the recycling process of mitochondria. These findings remained consistent at both DIV40 and DIV90. Notably, the overexpression of NATO3 significantly restored both parameters of mitochondrial morphology at both time points (Fig.5A and B). These results indicate a role for Nato3 in maintaining mitochondrial health as previously shown in mice (Miozzo et al., 2022). Example 5 - NATO3 overexpression improves autophagic flux The A53T point mutation and resulting intracellular accumulation of α-Syn has been shown to disrupt the autophagy lysosomal pathway (Abeliovich & Gitler, 2016). We investigated the status of autophagy in hiPSC-derived DA neurons by staining for the autophagosome marker LC3-II and the cargo adapter protein P62. At DIV40, PD neurons expressing the control construct presented reduced levels of P62, while LC3-II levels were unchanged compared to the control neurons (Fig.6A and B, left panels). However, at DIV90, both P62 and LC3-II levels increased compared to the control, indicating a progressive accumulation of autophagy markers over time (Fig.6A and B, right panels). These observations suggest that defective autophagy becomes apparent in aged DA neurons carrying the A53T α-Syn mutation. Notably, the expression of NATO3 restored P62 levels to control levels in the PD line at both time points (Fig.6A). LC3-II levels were not altered by NATO3 expression at DIV40, the time point at which no significant difference was present between PD and control cells. Whereas at DIV90, NATO3 overexpression significantly reduced the LC3-II levels in the PD line, below the levels of the control cells expressing the GFP control. NATO3 overexpression also reduced the LC3- II levels in the control cells (Fig.6B). Taken together, these results indicate that NATO3 overexpression stimulates autophagic flux, thereby restoring defective autophagy in PD neurons. Example 6 - Nato3 overexpression prevents locomotor deficits and degeneration of substantia nigra dopaminergic neurons in mouse models of Parkinson’s disease Methods Mouse strain. Both male and female C57BL / 6 wild-type mice were used in all the experiments. Mice were maintained in rooms with controlled 12 h light / dark cycles, temperature between 23–24 °C, and humidity of 47–61%, with food and water provided ad libitum. Mice were housed at a maximum of five animals per cage in individually ventilated cages. All experiments were conducted in accordance with the Institutional Animal Care and Use Committee of the University of Geneva and with permission of the cantonal authorities (Permit No. GE / 266B). Behavioral tests. Three-months-old mice were evaluated for posture control and coordination using a pole test as previously described (Matsuura et al., 1997) with minor modifications. All behavioral tests were performed during the light cycle. Mice were allowed to habituate to the behavioral room for at least 45 min before each test. Behavioral equipment was cleaned with 70% ethanol after each test session to avoid olfactory cues. Mice were first trained 4–6 times by placing the animal head-down on top of a vertical pole (diameter: 1 cm, height: 55 cm) and letting it descend. Then, animals were trained 3–4 times in the regular turning and descending procedure, by placing the animal head-up on top of the pole. For the actual test, mice performed nine trials (three trials per day for three consecutive days) with an interval of at least 5 min between trials and were recorded with a video camera. The time to orient downward (T-turn) and the total time to turn and descend the pole (T-tot) were measured, with a maximum duration of 30 sec. When the mouse was not able to turn downward and instead dropped from the pole in a lateral body position, a value of 30 sec was assigned. The average of the nine trials was used as the final score. Stereotactic injection. Three-months old mice were anesthetized with isoflurane and unilaterally injected in the substantia nigra (right hemisphere) with 2μL of a suspension of the different vectors used. The titer in the vector suspension of AAV2 / 7-pgk1-^Syn- WPRE vector (3.4 x 1013vector genome (VG) / mL), encoding wild-type human ^- synuclein protein, was determined by real-time polymerase chain reaction on total DNA derived from HEK293T cells, 48h after infection with AAV2 / 7. The titer in the vector suspension of AAV2 / 7-pgk-empty-WPRE vector, used as control for comparison, was 2.6 x 1013VG / mL. The injected vectors dose was set at 1.5 x 109VG. For Nato3 overexpression, the titer in the vector suspension of AAV6-pgk-Nato3-WPRE vector is 5.4 x 1011VG / mL. The injected vector dose was set at 5.4 x 1011VG. PBS only was also injected and used as a control. All virus production was performed by Bernard Schneider and his team (EPFL-Lausanne, Switzerland). The vectors were injected using a standard stereotaxic procedure according to the following coordinates : -3.0 mm (anteroposterior), - 1.3 mm (mediolateral) and -4.7 mm (dorsoventral). MPTP injections. MPTP hydrochloride (Sigma, Ref#5063820001) was administrated by intraperitoneal (IP) injections (20mg / Kg body weight in saline solution) for 5 consecutive days. Control mice were injected with saline only. Mice were euthanized 1 month after the first IP injection. Brain collection. Prior to intracardiac perfusions, animals were anesthetized with pentobarbital. Heparin was first perfused in the left ventricle of the heart to prevent blood coagulation, followed by 10% PFA perfusion. Subsequently, brains were collected and fixed for 3 additional nights in 4% PFA before being stored in 1X PBS at 4°C. Brain sectioning. Brains were embedded in 6% low melting agarose and 40 μm-thick sections were cut using vibratome. Sectioning of the entire striatum and substantia nigra was performed and each slice was put in 1X PBS and stored at 4°C in 24-well plates. Immunofluorescence. The number of DA neurons in the substantia nigra were measured by immunofluorescence for tyrosine hydroxylase (TH). Three sections of striatum and every fourth section covering the entire substantia nigra were included in the staining procedure and analysis. Slices were incubated in a blocking solution (BSA 4%, NGS 4%, Triton 0,1% in 1X PBS) for 30 min, then incubated 48h at 4°C with the primary antibody solution (Anti-TH, made in rabbit 1:1000 (Sigma Ref#ab152) in Tween 0,1%, NGS 4% in 1X PBS). After washing (Tween 0,1% in 1X PBS), slices were incubated with the secondary antibody solution (Goat anti-rabbit coupled with 488 fluorochrome 1:1000, in Tween 0,1%, NGS 4% in 1X PBS) overnight in the dark at 4°C. Finally, after two washings, nuclei were stained with DAPI (5µM) for 30min. Brain slices were mounted with Vectashield on microscopy slides. Microscopy and image analysis. Scanning of immunostainned brain sections was performed by using a Nikon AX confocal microscope as previously described (Berman et al., 2011). Total number of DA neurons detected by anti-TH immunofluorescence was automatically assessed using the QuPath software. Substantia nigra of each hemisphere were analyzed separately. Statistics. Data were analyzed using the GraphPad Prism software (Version 10.4.0). Experiments were performed on both males and females. Data were first submitted to the outliers test (ROUT Method with Q=1%). Identified outliers were removed for subsequent analysis. Normal distribution of the data was then assessed using an Anderson-Darling test. When the data passed normality test, parametric tests were performed: two-tailed unpaired t-test. When the data did not pass normality test, non-parametric tests were performed: two-tailed Mann-Whitney test. A p<0.05 was considered statistically significant and is presented as follows: *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. Results and discussion To investigate if Nato3 expression prevents pathological characteristics of Parkinson’s disease (PD) in vivo, particularly the loss of substantia nigra DA neurons and motor dysfunction, we generated AAV6-pgk-mNato3-WPRE (AAV-Nato3), which expressed mouse Nato3 (mNato3) under the control of the phosphoglycerate kinase 1 (pgk1) promoter (SEQ ID NO: 17). The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE, SEQ ID NO: 18) was added before a polyadenylation signal to increase the expression levels of mNato3. The viral vectors were produced at the Bertarelli Foundation Gene Therapy Core Facility of EPFL. We focused on two well-established mouse models of PD: 1-metyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) model and a model for ^^-synuclein (^^Syn)-linked PD. MPTP is a mitochondrial toxin that causes selective loss of DA neurons (Meredith & Rademacher, 2011). Among various protocols of MPTP treatment, we verified that 20 mg / Kg MPTP IP injections once a day for 5 consecutive days effectively induced nigral DA neuron loss but did not inflict motor dysfunction. To test the effect of Nato3 overexpression in the MPTP model, AAV-Nato3 was stereotactically injected into one side of the substantia nigra of 3-month-old mice. One week after the stereotactic injection, the mice received IP injections of MPTP 20mg / Kg daily for 5 consecutive days. The number of DA neurons in the substantia nigra were analyzed one month after the MPTP treatment. We found that MPTP IP injection resulted in a loss of DA neurons in the substantia nigra as expected. Importantly, AAV-Nato3 injection effectively suppressed the MPTP-induced DA neuron loss (Fig.7). It has been shown that AAV2 / 7 vector-mediated overexpression of ^^Syn in the substantia nigra induces a progressive loss of DA neurons in the injected site accompanied by the locomotor impairments (Oliveras-Salvá et al., 2013). We generated an AAV2 / 7 vector expressing ^^Syn under the control of the neuron-specific synapsin 1 (syn1) promoter (AAV2 / 7-syn1- ^^Syn WT-WPRE; AAV-^^Syn) and a control vector that did not express any gene (AAV2 / 7-syn1 -WPRE) and verified that stereotactic injection of AAV-^^Syn but not the control vector led to nigral DA neuron loss and locomotor dysfunction. To test the effect of Nato3 overexpression on PD-related pathological characteristics in this model, AAV2 / 7-^^Syn was stereotactically injected into one side of the substantia nigra, with or without co-injection of AAV-Nato3, in 3-month-old mice. Two months post- injection, locomotor behavior was analyzed using a pole test, and DA neurons in the substantia nigra were assessed with anti-TH immunostaining. We found that the locomotor impairments caused by AAV-^^Syn were significantly inhibited by the co-injection of AAV-Nato3 (Fig.8 and 9). The p-value of p=0.058 as shown in Fig.9c is the correct value. In addition, AAV-Nato3 co-injection prevented the loss of DA neuron in the substantia nigra induced by AAV- ^^Syn (Fig.10). Taken together, these results indicate that Nato3 overexpression can protect nigral DA neurons from degeneration, and also prevent locomotor deficits. Sequences of the invention SEQ ID NO: 1: DNA sequence encoding wild-type human NATO3 transcript, 5’ and 3’ UTR in lowercase, coding sequence in upper case. accagcaacctcgcccctccctgcggaaaaccgatgagaggcagggccaa gccgaagcgATGGCGGCCTATCCGGAGAGCTGCGTGGACACTACGGTGCT GGACTTCGTCGCAGACCTGTCCCTGGCCTCCCCGAGACGCCCTCTCCTCT GCGACTTCGCACCCGGGGTCTCCTTGGGGGACCCAGCCCTTGCGCTCCGA GAGGGAAGACCCAGGAGGATGGCGCGGTTTGAAGAGGGGGACCCAGAAGA AGAGGAGTGCGAAGTGGACCAGGGGGACGGAGAAGAGGAGGAGGAAGAGG AGCGCGGAAGAGGTGTCTCCCTATTAGGCCGCCCCAAGAGGAAAAGGGTG ATCACCTACGCCCAGCGCCAGGCCGCCAACATCCGCGAAAGGAAGCGGAT GTTCAACCTCAACGAGGCCTTTGACCAGCTGCGGAGGAAGGTGCCCACGT TTGCTTACGAGAAAAGGCTGTCCCGGATCGAGACCCTCCGCCTGGCCATC GTCTATATCTCCTTCATGACCGAGCTCTTGGAGAGCTGTGAGAAGAAGGA AAGCGGCTGAgcctggtgtggagagtctgcccttcctcgtctggtagtgc tggggtgtgtcaggaccgggcactgggtgaggctaaaggg SEQ ID NO:2: wild-type human NATO3 amino acid sequence MAAYPESCVDTTVLDFVADLSLASPRRPLLCDFAPGVSLGDPALALREGRPRRMARFEEG DPEEEECEVDQGDGEEEEEEERGRGVSLLGRPKRKRVITYAQRQAANIRERKRMFNLNEA FDQLRRKVPTFAYEKRLSRIETLRLAIVYISFMTELLESCEKKESG

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Claims

CLAIMS 1. An agent that increases Nato3 expression and / or activity for use in a method for preventing or treating a neurological disorder.

2. The agent according to claim 1, which comprises an expression construct comprising a promoter operably linked to a nucleic acid sequence encoding an agent that increases Nato3 expression and / or activity, or a vector comprising said expression construct.

3. The agent according to claim 2, which is a viral vector, optionally an adeno associated virus (AAV) vector or a lentiviral vector.

4. The agent according to claim 3, which is a lentiviral vector.

5. The agent according to any one of the preceding claims, which is a cell comprising an agent that increases Nato3 expression and / or activity, an expression construct comprising a promoter operably linked to a nucleic acid sequence encoding an agent that increases Nato3 expression and / or activity, or a vector comprising said expression construct.

6. The agent according to any one of the preceding claims, which comprises a Nato3 polypeptide or a nucleic acid sequence encoding a Nato3 polypeptide.

7. The agent according to claim 5 or 6, which is a dopaminergic neuron or a dopaminergic progenitor, optionally derived from a patient with a neurological disorder.

8. The agent for use according to any one of the preceding claims, wherein the neurological disorder is a neurodegenerative disorder, optionally characterised by overexpression and / or aggregation of α-synuclein and / or the presence of Lewy bodies.

9. The agent for use according to claim 8, wherein the neurodegenerative disorder is Parkinson’s or Alzheimer’s disease.

10. The agent for use according to any one of the preceding claims, wherein: administration of the agent prevents or reduces one or more of the following phenotypes associated with the neurological disease: neurodegeneration of dopaminergic neurons in the substantia nigra, synapse degeneration,mitochondrial dysfunction, defective autophagic flux, oxidative stress, α- synuclein overexpression and / or aggregation and / or Lewy bodies.

11. The agent for use according to any one of claims 1-9, wherein administration of the agent prevents or reduces loss of dopaminergic neurons of the substantia nigra and / or prevents or reduces locomotor deficits or motor dysfunction.

12. The agent for use according to any one of the preceding claims, which is: (i) administered to a patient comprising an overexpression and / or aggregation of α-synuclein and / or Lewy bodies, optionally in dopaminergic neurons; and / or (ii) administered to a patient comprising an α-synuclein mutation causing overexpression and / or aggregation of α-synuclein, optionally A53T.

13. An expression construct comprising a promoter operably linked to a nucleic acid sequence encoding an agent that increases Nato3 expression and / or activity, optionally wherein said nucleic acid sequence encodes a Nato3 polypeptide; or a vector comprising a said expression construct, optionally a viral vector, optionally wherein the viral vector is an adeno associated virus (AAV) vector or a lentiviral vector; or a cell comprising a said expression construct or vector.

14. A dopaminergic neuron or a dopaminergic progenitor comprising an expression construct comprising a promoter operably linked to a nucleic acid sequence encoding an agent that increases Nato3 expression and / or activity or a vector comprising said expression construct; optionally wherein said vector is a viral vector, optionally an adeno associated virus (AAV) vector or a lentiviral vector; optionally wherein said nucleic acid sequence encodes a Nato3 polypeptide.

15. A non-human organism comprising in its genome an expression construct comprising a promoter operably linked to a nucleic acid sequence encoding an agent that increases Nato3 expression and / or activity or a vector comprising said expression construct; optionally wherein the non-human organism is a rodent, optionally a mouse or rat; optionally wherein said nucleic acid sequence encodes a Nato3 polypeptide.

16. A method of screening for a compound that improves dopaminergic function, comprising contacting a dopaminergic neuron as defined in claim 14 or a non- human organism as defined in claim 15 with a compound, and monitoring said neuron for improvement of one or more parameters of dopaminergic function; optionally wherein the one or more parameters of dopaminergic function are selected from neurite length and / or complexity, mitochondrial function, autophagic flux, and α-synuclein expression and / or aggregation; optionally wherein the dopaminergic neuron is derived from a patient at risk of, or having a neurological disorder, optionally Parkinson’s disease.

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