GENES WITH RESTRICTED EXPRESSION IN MESENCEPHALIC DOPAMINERGIC NEURONS

Abstract: The invention relates to the field of neurology, psychiatry, neuropathology and neuropharmacology, more specifically to neurological and psychiatric disorders such as Parkinson's disease and tardive dyskinesia, and psychiatric disorders such as schizophrenia, addiction and affective disorders, like manic depression, and other disorders related to malfunctioning of the mesencephalic dopaminergic (mesDA) system. The invention provides an isolated and/or recombinant nucleic acid or a specific fragment, homologue or derivative thereof, corresponding to a gene with restricted expression in mesencephalic dopaminergic neurons, wherein said gene is related to neurological and psychiatric disorders such as Parkinson's disease, tardive dyskinesia, manic depression and schizophrenia.

Description: Title: Genes with restricted expression in mesencephalic dopaminergic neurons . The invention relates to the field of neurology, psychiatry, neuropathology and neuropharmacology, more specifically to neurological disorders such as Parkinson's disease and tardive dyskinesia, and psychiatric disorders such as schizophrenia, addiction and affective disorders like manic depression, and other disorders related to malfunctioning of the mesencephalic dopaminergic (mesDA) system. The mesDA system consists of groups of neurons located in the substantia nigra (SN) and ventral tegmental area (VTA) of the mesencephalon of all mammals. These neurons project axons towards a number of forebrain areas, including cortical, striatal and limbic brain areas, where dopamine (DA) is acting as neurotransmitter regulating a variety of brain functions. Brain functions regulated by mesDA neurons include the control of movement and behaviours. A number of neurological and psychiatric disorders are implicated as caused by a malfunctioning mesDA system. Among these neurological disorders one finds extra-pyramidal motor disorders such as degeneration of mesDA neurons which is the cause of Parkinson's disease. Depending on which subset of neurons is affected the clinical symptoms of Parkinson's disease patients may differ. During the course of Huntington's chorea the striato-nigral pathway degenerates causing motor disturbance and uncontrolled movement. Also, the mesDA system is involved in tardive diskinesia, which is a movement disorder manifested by involuntary movements, often seen as side effect of anti-psychotic drug treatment. Furthermore, psychiatric disorders such as schizophrenia are known, where a fundamental, developmental malfunctioning of the mesDA system is a proposed aetiology. Manic-depression is associated with the mesDA system as well; just as well as addictive behaviours, most importantly drug addiction, are linked to the activity of the mesDA system. Malfunctions of the mesDA system may be of a various nature; for example its development can be affected resulting in a inappropriate innervation of target brain areas or a failure to synthesise dopamine . Furthermore, malfunctioning can be related to the maintenance of the mesDA system; an intrinsic drive of gene expression may be necessary to maintain functional integrity and protect against degradation by radicals or other noxious stimuli. Neurological disorders affecting movement related to the jnesDA system are relatively well understood, in contrast to psychiatric disorders related to the mesDA system. However, considering the relative importance of this group of disorders, a need exists to be able to better diagnose and treat these diseases. For example, Parkinson's disease is a degenerative neurologic disorder that effects several millions of people. There is no known medical treatment that stops or reverses the dopaminergic neuronal degeneration that causes the symptoms. Investigators are searching for new surgical treatments that may provide better control of symptoms for longer periods of time. Re-innervating the basal ganglia with dopaminergic neurons is the theory behind (foetal) neural tissue transplantation. However, much research is still needed before foetal neural tissue transplantation can be offered as a therapeutic option for patients with Parkinson's disease. Neuropathological diagnosis of Parkinson's disease is currently based on examination of post-mortem brain sections macroscopically and microscopically after HE staining looking for Lewy bodies, or by immunohistochemistry for tyrosine hydroxylase, an enzyme critical for catecholamine (dopamine, noradrenalin, adrenalin) production. A major disadvantage is that an accurate diagnosis can only be made after the patient has died; there is currently no other way of diagnosing this disease in an earlier phase, except by a diagnosis on more- or-less well understood clinical and neuropsychological symptoms. As a consequence, patients are often diagnosed late in the course of the disease. An additional disadvantage is the presence of tyrosine hydroxylase in other neurons than those affected by Parkinson's disease, which can lead to faulty diagnosis if the mesDA neurons are not accurately localised. Furthermore, a body of evidence exists that Parkinson's disease may be genetically predisposed, whereby several different phenotypes have been associated with autosomal (dominant as well as recessive) or X- or Y-linked genetic causes; Lewy body Parkinson's disease is one of these examples. It seems possible, indeed likely, that parkinsonism is heterogeneous, with some mendelian forms of the disease. A genetic link between patients with Parkinson's disease and patients with manic depression has been reported as well. Yet other causes of Parkinson's disease are thought to be intoxications with toxic substances such as meperidine and other neurotoxic xenobiotics, and metal ions such as manganese . Only a limited number of animal models are available for studying Parkinson's disease and other neurological and psychiatric disorders related to the mesDA system, mainly because the pathogenesis of the disease is badly understood. Progressive postnatal depletion of dopaminergic cells has been demonstrated in Weaver mice, a mouse model of Parkinson's disease associated with homozygosity for a mutation in a gene encoding a part of a potassium channel . However, this model bears no relevance to human disease, said genetic defect is not found with human patients. Another, mesDA cells injected with 6-hydroxydopamine (6-OHDA) in the corpus striatum or sybstantia nigra/VTA of the rat brain. This model, however, depends on the creation of irreversible lesions in the mid-brain of the rat, creating more or less serious losses of dopaminergic neurons. No rat can be considered having been given the same lesion, consequently, this model is inaccurate, and gives no insight in development of the disease. Another suggested model entails the use of Nurrl- deficient mice. Nurrl is expressed in the mid-brain of mice during embryonic development, but expression is not restricted to this area. Nurrl activity during embyronic development is thought to be part of a very generalised signaling mechanism that cells use. Nurrl knock-out mice fail to develop mesDA neurons, making them putative candidates for studying neurological disorders related to the mesDA system, such as Parkinson's disease. However, Nurrl expression is not restricted to mesDA neurons but is also part of an overall signalling mechanism that cells use. It is evidently needed in other organ systems more central to the viability of the mice involved. Homozygous Nurrl knock-out mice die within two days after birth and are therefore not available as an animal model. Although heterozygous Nurrl knock-out mice show reduced dopamine levels, they do not show apparent histological or behavioural abnormalities, again making them unsuited for use as an animal model. The same is seen with knock-out dopamine deficient mice wherein the tyrosine hydroxylase gene was disrupted, these die within two weeks after birth, probably also because the tyrosine hydroxylase gene is essential for viability of many more cells and tissues. Yet another possible factor involved with dopaminergic neurons, GDNF (glial cell-line derived neurotrophic factor) has also been studied. Although GDNF is a potent survival factor for among others dopaminergic neurons, GDNF knock-out mice have no lesions in the mesDA system but fail to develop kidneys and have lesions in the enteric nervous system instead. Until now, mesDA related genes that for example can be used to create an animal model to be used to study neurological disorders, are not available. The invention provides a mesDA related nucleic acid and means and methods which allow studying neurological disorders related to the mesDA system. The invention provides methods and means of diagnosing such neurological disorders and suitable animal models wherein the mesDA system and its development can be studied. The invention provides an isolated and/or recombinant nucleic acid or a specific fragment, homologue or derivative thereof, corresponding to a gene with restricted expression in mesencephalic dopaminergic neurons. Where in this application the definition 'nucleic acid' is used, both RNA and DNA, in single or double-stranded fashion, and nucleic acid hybridising thereto is meant. Also, when the definition 'nucleic acid' is used, a specific fragment, homologue or derivative of a nucleic acid provided by the invention is meant as well. The meaning of 'specific fragment', 'homologue' and 'derivative' is clear to those skilled in the art. 'Specific fragment' meaning a nucleic acid or part thereof that is functionally or structurally related to or hybridising with a distinct nucleic acid or fragment thereof. 'Homologue' meaning a related nucleic acid that can be found with another gene or with another species. 'Derivative' meaning a nucleic acid that has been derived by genetic modifications, such as deletions, insertions, fusions and mutations from a distinct nucleic acid. A nucleic acid provided by the invention corresponds to a gene or its gene product (nucleic acid and/or protein) that forms a part of the regulatory cascade for the embryonic development and the maintenance in adulthood of the mesencephalic dopaminergic neuron (mesDA) system. A characteristic feature of a regulatory cascade concerns the fact that a variety of genes and gene products act in concert. These genes act, often within a certain time span, some synchronously, others sequentially, to activate tissue and/or cell specific gene expression and differentiation, thereby allowing embryonic cells of yet unspecified or partly specified nature to further differentiate into a more mature cell tissue and/or organ. Such genes for example encode nuclear hormone receptors or homeodomain proteins, which are transcription factors that regulate major developmental processes. These factors depend on interactions with each other and with other genes and proteins, in regulating a cascade of developmental events that contribute to the further differentiation of as yet undifferentiated or partly differentiated embryonic cells. The invention provides an isolated and/or recombinant nucleic acid or a specific fragment, homologue or derivative thereof, corresponding to a gene that is functional in the regulatory cascade leading to the differentiation of mesDA neurons. The regulatory cascade is involved in restricting the future nature of the embryonic cell, whereby the cell is loosing its general, unspecified nature and gains specific characteristics and properties. This differentiation process is regulated by a cascade of gene and gene product interactions, whereby generally interactions that come in a later phase (downstream) are of a more restricted nature then those interactions that occur earlier (upstream) . Upstream activators or regulators in general show a broad expression, whereby activity can be detected in a broad range of (as yet undifferentiated) cells. A nucleic acid that is corresponding to a gene with restricted expression corresponds to a gene which activates or regulates a later phase in a regulatory cascade; whereby its expression (insofar as it is related to the development of that specific cell type) is restricted to one or a few specific cell types in that phase of development. Expression of upstream regulators is in general less restricted than the expression of downstream regulators . The invention provides a nucleic acid corresponding to a gene that is involved in the regulatory cascade of the embryonic development of the mesDA system. Said gene is involved in the regulatory cascade, activating a programme for mesDA-specific gene expression and differentiation. An example of a nucleic acid (of which a corresponding sequence is given in figure 5) provided by the invention relates to a Ptx3 gene. The invention further provides a method to identify a gene related to the regulatory cascade involved in the development of the mesDA system. One example of such a -" method according to the invention is using mutant or even transgenic cells or animals (54; 55) . By analysing mesDA cellular markers in for example a transgenic or mutant cell or animal provided by the invention (for example an animal expressing ectopic Ptx3 and/or a functional mutant of Ptx3 or a knock-out mutant) we can examine what the influence of said gene on the expression of other mesDA genes is. According to the invention, changes in the expression patterns are observed by making libraries of the expressed mRNAs which can be subtracted from each other. This yields the specific expressed mRNA in the wild type cell or animal, as compared to the transgenic or mutant cell or animal. A similar approach is the differential display where one performs PCR on both species mRNA and compares the results in respect to each other. The bands that appear specifically in the wild type, as compared to the mutant or transgenic cell or animal, represent a nucleic acid that is under control of for example Ptx3. This specific PCR product is sequenced and analysed to further determine a nucleic acid sequence of a mesDA gene. Yet another example of a method to identify a gene related to the regulatory cascade involved in the development of the mesDA system entails the use of methods to study protein interaction in the regulatory cascade. According to the invention Ptx3 interacting proteins are identified. Methods studying such protein interaction are known in the art . One can for example use yeast two-hybrid screening or immuno-EMSA coupled to UV-crosslinking (see 44;45 ;46;47 ; ) . According to the invention the interactions of different domains of for example Ptx3 are studied in a yeast two-hybrid screen. This system is based on an approach that the protein of interest can interact with a library of other proteins. When the two proteins are interacting, a functional transcription activation signal is generated that expresses a marker in the yeast. These marker expressing yeast is isolated and amplified. From such clonal lines the plasmids are retrieved which produce the cDNA of the interacting protein, thereby generating the sequence of yet another gene involved in the regulatory cascade and restricted expression of the mesDA system. According to the invention protein-DNA-binding is determined by in-vitro UV-cross linking experiments during a DNA -binding experiment. Protein complexes are examined by Western analysis using specific antibodies for expected target proteins. Further studies of a gene related to the regulatory cascade of the embryonic development of the mesDA system are for example achieved by a method, such as expression studies during embryonic development, that are further explained in detail in the experimental part of this description. The invention provides an isolated and/or recombinant nucleic acid, or a specific fragment, homologue or derivative thereof, corresponding to a gene being related to neurological disorders such as Parkinson's disease and tardive dyskinesia and psychiatric disorders such as schizophrenia, addiction and affective disorders like manic depression. An example of a nucleic acid provided by the invention is a nucleic acid encoding (parts of) a Ptx3 gene or homologue which is expressed in neurons of the mesDA system of vertebrates . The invention further provides a method identifying a gene related to neurological disorders such as Parkinson's disease and tardive dyskinesia, and psychiatric disorders such as schizophrenia, addiction and affective disorders like manic depression. Expression of for example a Ptx3 gene in neurons ' of the mesDA system is maintained in adult individuals, whereas expression is reduced, enhanced or absent in neurons of the mesDA system of individuals having a neurological disorder related to dysfunctioning of neurons of the mesDA system. The invention provides a nucleic acid corresponding to a gene which is related to a homeodomain gene, preferably being a bicoid-related homeodomain gene, with restricted expression in the mesDA system and related to homeodomain protein- associating proteins and homeodomain-heterodimerizating partnbers. A example of a gene provided by the invention is given with the Ptx3 gene in the experimental part, wherein in figure 1 a (bicoid-related) homeodomain is depicted. Ptx3 is expressed in the mesDA system during development, where it activates a programme for mesDA specific gene expression and differentiation, and maintains its expression in healthy neurons of adult individuals . A preferred embodiment of the invention provides a nucleic acid according to the invention which gene is of vertebrate origin. In birds, the majority of L-DOPA- and DA-ir perikarya is, however, situated in the mesencephalic tegmentum, in the area ventralis of Tsai and in the nucleus tegmenti pedunculo- pontinus, pars compacta which are the avian homologues of, respectively, the ventral tegmental area and the substantia nigra of mammals. Also in amphibians the analog system is found in the mid brain tegmentum. A preferred embodiment of the invention provides a nucleic acid hybridising to (parts of) a nucleic acid with a nucleic acid sequence as listed in figure 5. In the experimental part, several examples are given of such a nucleic acid hybridising with a part of DNA of a Ptx3 gene of rat origin. For example, the invention provides a nucleic acid hybridising to a nucleic acid present in mouse embryonic tissue being expressed during embryonic development and showing restricted expression in neurons of the mesDA system, or hybridising to a nucleic acid with for example a genomic organisation as shown in figure 6. As yet another example, the invention provides a nucleic acid hybridising to a nucleic acid present in human brain ra 1 Φ d r-i ^ d P CJ xi Φ -H M rH J -H d d rd rd o 4-1 ra -H • CJ Cn υ ft d 4H 0 o CJ -H rl -H a Φ Φ . '1 4J X) a d d rd g rd T 0 TJ -H rH >i -H -H CQ d Φ 4-1 XI g rH rl ra rd ■ H rH rd Φ Φ CJ CQ rd A 4-1 Cn φ 0 XI rd >i o TJ rd TJ rl rd ft X ra ra n 4J -H a CQ υ Cn rd 0 d -H 4J -rl rH r Φ 3 d Φ ra rl Φ Φ a d d Φ Φ Φ -H d TJ -H d rH -H 4-> 0 υ e r-H r-H 0 XI d rd Φ > TJ Cn X! TJ rd υ -H rd rH A rl Cn -H Φ -H 0 A. υ 0 ra Q rd n d -H g r. 0 4H rl φ ft . Xl φ rH g rd X Φ rl O 4H - CQ rl φ TJ SH 0 ra -H Tj rH rd J -H σ. 0 rH O SH a φ d studies and sibling analyses on groups of (related) individuals tested both for allelic variation and for neurological disorders . Such allelic variation are found in humans by locating mutations, or other variations, in the Ptx3 gene in humans by using methods such as sequencing, restriction fragment length polymorphism, SSCP analysis and others that are available in the art (see for example 49; 50 ; 51 ;52 ; 53 ) . Cloning of cDNA or genomic DNA of patients, gives the opportunity to screen for mutations in patients that are affected in the mesDA system. By PCR cloning of the genomic DNA of the patients and sequencing it, mutations and variations are easily assessed. A more broader approach is to study restriction fragment polymorphisms. By this technique mutations and variations in the gene are found due to altered appearance of restriction analysis followed by Southern hybridisation of the genomic DNA. A more sensitive method is the single-strand conformation polymorphism. In this method variations in a given gene are detected by the altered characteristics of the single stranded DNA fragment spanning the gene of interest. The invention provides a method for determining expression of a gene using a nucleic acid according to the invention. A preferred embodiment of the invention is wherein said gene is related to neurological disorders or wherein said expression is determined in neurons. An example of a method provided by the invention is the detection of Ptx3 expression in the mesDA system of adult rat or human brain tissue, for example in neurons in the substantia nigra or ventral tegmental area, as further explained in the experimental part of the description. Another example provided by the invention is the detection of restricted Ptx3 expression in embryonic mouse tissue sections, for example in lens, sclerotome, tongue and brain tissue at various stages of embryonic development . The invention also provides a protein or peptide comprising an amino acid sequence encoded by a nucleic acid according to the invention. An example provided by the invention is a protein or peptide or fragment thereof comprising at least a part of a Ptx3 amino acid sequence as shown in the experimental part of the description, for example in figure 1. Yet another example provided by the invention is a fusion protein, for example of a part of GDNF and part of Ptx3 , created using cloning techniques as known in the art. For example, an DNA construct encoding such a fusion protein, which contains a part of the GDNF cDNA and a part of the Ptx3 cDNA fused together in the proper reading frame, is created by isolating both coding regions (by PCR) and cloning parts of them in the correct reading frame after each other. The resulting DNA fragment can be placed in a eukaryotic expression vector which contains the necessary signals to ensure a stable mRNA transcript. The protein is expressed in an eukaryotic cell-line and purified by use of standard purification methods. Yet another example entails prokaryotic expression of recombinant protein, for example Ptx3 protein, by cloning cDNA in a prokaryotic expression vector, which optionally fuses a highly expressed protein to for example Ptx3. A resulting (chimeric) protein can be produced in large amounts and purified. The resulting protein or peptide can be injected into an animal to raise antibodies. The invention provides a natural or synthetic antibody directed against a protein or peptide according to the invention. Antibodies (poly- or monoclonal) are produced as indicated above, or by other methods known in the art, for example by phage-display or related techniques whereby so- called synthetic antibodies are produced. A polyclonal antiserum (Ptx3-Abms01) raised in rabbit against a C-terminal portion of the rat Ptx3 protein fused to bacterial gluthathion-S-transferase has been obtained. Ptx3-Abms01 recognizes Ptx3 of rat, mouse and human. Ptx3 protein of these species are detected on Western Blots of biological X SH rH φ O ar Φ X! a TJ U 0 rd ra Φ CD T Tj -H CJ cn 3 0 'φ d ra rd SH φ Φ d ra Φ CM -rl d SH 0 Φ g 0 3 CQ rH 4J g g d rd 4-1 4-1 TJ rd d r-i U rd Cn 0 -H 3 rd CQ rd CJ rd d g 0 ft O O TJ 0 Φ SH 4J SH 0 CJ -H XI Tj Cn -H rH rd -H g O n r-l SH 4-1 fragment thereof is detected in cerebrospinal fluid. The invention for example provides a method to trace or detect mesDA degeneration in human CSF. Ptx3 cDNA is used as an exclusive marker for mesDA neurons by using parts of the DNA as in-situ hybridisation probes or use of the Ptx3 antibodies in immunological detection methods. Specific antibodies are also used to detect Ptx3 proteins in other material like CSF, by usage of immuno-precipitation. The presence of Ptx3 mRNA or protein in CSF originates from damaged mesDA neurones . For example, Ptx3 protein is detected in CSF of patients for diagnostic purposes by radioimmunoassay (RIA) or by Western blotting according to established principles. CSF of patients is obtained by lumbar punction. Aliquots of CSF are for example tested in a (radio) -immunoassay using a specific radioactive, fluorescent or enzymatic label, and for example recombinant Ptx3 protein as tracer and/or recombinant Ptx3 protein as standard, according to standard procedures. The detection of Ptx3 in CSF samples is for example based on the specific displacement of or competition with labelled Ptx3 , but other approaches for immunoassays are also known in the art. For example, by Western blotting a protein fraction of a concentrated CSF sample is separated according to size by polyacrylamide gel electrophoresis, transferred to a nitrocellulose or nylon membrane, and incubated with a pPtx3 specific antibody. The detection of Ptx3 in CSF samples is based on the specific staining of Ptx3 protein on the membrane . The invention also provides use of a method according to the invention to detect or diagnose a neurological disorder, such as Parkinson's disease and tardive dyskinesia and psychiatric disorders such as schizophrenia, addiction and affective disorders, like manic depression, or use of a method according the invention to test or develop specific medication for the treatment of a neurological disorder. Diagnosing a disease or developing medication for it is possible when a pathogenetic mechanism has been elucidated Φ rd 4H d Q d d xi 0 d . . Φ -H ■ H CQ d 0 0 4J rd d rd ra 0 H Φ SH 0 -H Φ -H '1 -rl φ ro υ rH SH ra ft > . TS SH - d Φ -H 4-1 Xl CQ • d SH rH X H 4 0 Φ g rH rH Φ rd d rd Φ 4H 4-1 d 4-1 ra d -rl 0 CQ A 4-1 Q φ t rd TJ rH rd d 4J >. 4-> 0 Φ Φ P. SH J " r> 1 > f φ d If) φ φ 0 3 rd 53 ■ rl SH X φ rd H υ A ra rH rd -H xi 4-1 X! SH φ VO > 4H SH ■ rl TJ SH υ ft P 0 0 φ Φ > υ 1 -H φ rH rd 4-1 4J e ra t> d -H Φ Xl 4J Φ rH & g rd X Φ SH 0 &. • rH rd g -H d rd CJ -H d Φ cn ra d rd rl 4J rd 0 4J . rH rH rd -H CJ Φ ft CQ Φ υ Cn φ φ . rd 1 ra rH .. -H d ro ra l XI m A. A. 3 rd rH rd - SH υ d ,1H X -rl 4J d CJ CJ ^ S xl Φ -H Φ d 0 -H Φ d 4-1 A ϋ d A. . -H 4H 0 O -H 4J 4-> υ ra d r-l 0 ft Cn -rl d CM EH -H rd d rd υ d d 4H d H υ CJ Φ ' 0 & -H Ϊ 0 ra rd -H g -rl υ 0 0 0 d A; 0 Al rH rd d Φ rd d -rl g 4J «Λ Cn 4J that either increases or decreases for example Ptx3 or dopamine activity in the mesDA system. Alternatively, it is possible to determine how toxins effect Ptx3 activity and identify a drug or pharmaceutical composition that counteract that toxicity. Furthermore, it is for example possible to find a pharmaceutical composition that boosts or restores Ptx3 activity in failing nerve cells which will delay or prevent the onset of Parkinsonian symptoms . Also the invention provides use of a cell according to the invention for the treatment of a neurological disorder, such as Parkinson's disease and tardive dyskinesia, and psychiatric disorders such as schizophrenia, addiction and affective disorders, like manic depression. Cells, such as undifferentiated nerve cells of various origin (foetal or porcine cells are a possibility) can be transfected with for example (parts of) Ptx3 nucleic acid, optionally comprising additional nucleic acid derived of another gene, such as the GDNF gen. Such cells are converted in dopamine producing cells that can be used to replace those that are for example damaged by Parkinson's disease. The invention provides a pharmaceutical composition comprising a protein or peptide according to the invention and its use for the treatment of a neurological disorder, such as Parkinson's disease and tardive dyskinesia, and psychiatric disorders such as schizophrenia, addiction and affective disorders, like manic depression. An example of such a pharmaceutical composition is a composition comprising a protein or peptide or fragment thereof comprising at least a part of a Ptx3 amino acid sequence as shown in the experimental part of the description, for example in figure 1, or a fusion protein, for example of a part of GDNF and part of Ptx3. In addition, the invention provides a pharmaceutical composition comprising an expression vector according to the invention, and its use for the treatment of a neurological disorder, such as Parkinson's disease and tardive dyskinesia, and psychiatric disorders such as schizophrenia, addiction and affective disorders like manic depression. An example of such a pharmaceutical composition is a composition comprising a a viral vector or a targeting vector specifically directed at a target locus in a genome of an experimental animal or human. Such a vector allows in vivo "" targeting of other proteins to mesDA neurons by using regulatory regions or promotor regions of for example the Ptx3 gene . A viral vector can be constructed for example for in vivo viral gene transfer in patients. Viruses engineered to transfer genes to in vivo tissues (e.g. adeno, AAV, retrovirus) are modified for expression of for example Ptx3 protein by inserting a cDNA of Ptx3 or Ptx3 -derived mutant. A pharmaceutical composition comprising a vector virus according to the invention is for example delivered locally in the substantia nigra of patients for example by micro- cannulation and injection. The invention is further explained in the experimental part which cannot be seen as limiting the invention. Experimental part Nuclear hormone receptor genes or homeodomain genes specify cell fate and positional identity in embryos throughout the animal kingdom. The homeodomain is a DNA binding motif which is characteristic and strongly conserved among the homeodomain proteins. The specificity of homeodomain binding to its target is based on distinct DNA binding properties of the homeodomain sequence and assembly with other proteins . Protein-protein interactions can involve both specific residues within the homeodomain itself and other regions of the protein, as has been suggested for the regions immediately C- and N-terminal to the homeodomain. The Ptx proteins are found to be most closely related to the Caenorhabdi tis elegans Unc-30 protein. Drosophila orthodenticle (Otd) and its murine homologs Otxl and Otx2 and the Paired-like proteins Drgll, al , Cart-1, Prx-1, Prx-2 and Chx-10. The Paired-like proteins were found to share a 14 amino acid motif located 3 ' of the homeodomain, with the proteins which may represent a DNA binding element or a site of protein-protein interaction playing a role in the specificity of the homeodomain protein function. Paradoxically, although each has a specific function in vivo, the in vi tro DNA-binding specificity of the proteins they encode are overlapping and relatively weak. Often, cofactors are needed to increase the specificity of binding of these proteins . The patterning of the developing mammalian brain is thought to involve cascades of signalling molecules and transcription factors, but the mechanisms for generation of distinct neuronal cell types during terminal differentiation are still largely speculative (1,2). Yet, the specification of individual neuronal phenotypes underlies the assembly of neural circuits essential for brain function. The mesDA system consists of a limited set of neurons that are well- defined anatomically and functionally (3-5) . Their specific degeneration in Parkinson's disease reveals their functional properties in control of behaviour and movement as well as a unique vulnerability (6-10) . In a search for homeobox genes associated with a unique neuronal lineage, we isolated a novel cDNA, encoding a bicoid related homeobox gene Ptx3 , a member of the Ptx subfamily (11-14) . METHODS AND MATERIALS Cloning of Ptx3 gene transcripts. Poly A+ RNA from hypothalamic fragments of the adult rat brain were subjected to reverse transcriptase/PCR with primers based on brain expressed homeobox genes: upstream: 5'- GMRSCGMSAVMGSACMMBCTTYAC -3', downstream: 5'- TGGTTYMRVAAYCGYHGMGCMARRTG -3*. The annealing temperature was XI - ^ ^ -*. Φ CQ d 4J + . d r-l 4J -rl -H ra 4J , X , ^ -rl r-l -rl Φ -H g CN S 4H ' Φ EH ] X d SH φ φ TJ -H Al 0 rH X d TJ Xl . rd -H rd 3 rH U -rl φ υ SH 0 4J . -H φ t 4J -U ~ X) ft 2 cn 4H ^ - ^ Φ 4J g d 4H SH o rH rd X! IΛ 3 d XI 3 rd rd J o K ^ SH CJ SH Φ H 4-1 CQ SH -H . TJ CQ φ n rH 4H SH -rl Φ ra 1 d rd d ,. d rd 0 -H ^ -_. 4J o CQ O -rl SH . o . — Tj '-rl SH Φ XI : . ra rd Φ xl d SH rH Ti >. 0 CQ -rl d φ co CQ d • 4H Xl 3 Xl 4J — • 3 ^ r H 4-1 -H ft xi rd d XI rH 4-1 Φ 4-1 -rl Xl 4H < d rd CQ Tj 0 4J 4-1 d EH d co ft Φ 4-1 -H -H CQ rd d ft 4-1 Φ 0 IΛ -H > d -H CJ -H Φ Φ -H vO g Φ 0 4-1 Φ 0 4J XI TJ 3 > Φ φ -H 0 SH »• ra Φ 3 ra ra -H 4J CQ 3 0 • H SH rd > d ■ rl Tj φ ra 5>ι rH rd d rd ra rd "? d 0 -H ra ra Φ SH S X φ ra 4J -H ro X 4-1 Pi 00 C w . SH X 4H rd 4H CQ d Φ Φ cn d P Φ ,. — , d 4-1 CJ Φ d 4H Φ d ■ rl 00 > 4-1 -rl , 0 Xl ra SH d tn X 0 TJ ,--_. TJ rd 4H 4J CQ g J . I CQ 4J Φ rd SH 4-1 A d rH 4-1 VO Φ Φ Xl o rd rH rd d Φ φ TS 0 rd 4-1 Φ CM rd rd rd . CQ CN Xl > x> rd Ss rl 4-J 5*1 4-J Φ rH rH XI 4J Xl d φ tn SH ^ 4-1 SH SH d SH φ φ Tj CQ IΛ Φ d J rd 4-1 rd 4J -H SH Φ rH ^ - ^ 4J -H -H in Φ 3s Φ 0 4J X! ra Φ 4-> d 5*ι e φ 3 g A ft -rl g Φ 4-1 rd d d Cn 4H o CQ 0 ι -H d 4-1 4-1 3 4J rd ra rfl P ra Φ g Cn d ft 0 rH Φ Φ TJ Φ Xl 4J 4H 0 φ J d rd SH rd Φ ft ft rd Φ Xl 4-J Xl 4J -H S rH rd SH O & g Φ 4J S 5>ι rd d XI SH Φ rd rd > rd d d d .. 0 Φ Φ TS Xl cn Φ TJ Ss TS ■ rl -rl o d φ XI SH Φ rd 0 ra υ SH d 0 d — - TS d 0 rd 3 0 rd rd xl Φ 0 rd XI rd Φ SH - Φ IΛ g oo SH rd cn rd m SH U Φ g ra Xl Φ rd g CJ TJ Ti rd tn 3 SH TJ TJ d rd d Φ g Φ u SH 0 d Φ rd SH 3 O 5* rd Xl φ XI d T ) d rd '— cn - (40) . Susceptibility to these disorders is thought to be predisposed prenatally. The identification of genes controlling developmental mechanisms of mesDA neurons can provide new insights in the aetiology of these disorders. Ptx3 is a candidate gene involved in such mechanisms. 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Rodriguez, I., Araki, K. , Khatib, K. , Martinou, J.C. & Vassalli P. (1997), Dev. Biol. 184, 115-121. 61. Bittner, H.B., Chen, E.P., Milano, C.A., Lefkowitz, R.J. & Van Trigt, P. (1997), J. Mol. Cell. Cardiol . 29, 961-967.; 62. Khillan, J.S. & Bao, Y. (1997), Biotechniques 22, 544- 549. 63. Tsien, J.Z., Chen, D.F., Gerber, D., Tom, C, Mercer, E.H., Anderson, D.J., Mayford, M. , Kandel, E.R. & Tonegawa, S. (1996), Cell 87, 1317-1326.). 64. Semina, E.V., Reiter, R.S. and Murray, J.C. (1997) Hum. Mol. Gen. 6, 2109-2116. Φ 4J CQ rd Φ CN • d SH 4J SH Xl cn X -- ^ Φ 0 d O 4-> XI B d 4-1 00 g 4-> rd J ra 4H rH Ti rH d 4J -H CM 1 CN ra Φ CJ 4-1 -H Ss rd d 3 -H -rl Ti Ti ' — Ti -H rH rd 3 0 0 Φ -H 00 rd Ti rd Ss rH 5 l rd Ti -H Φ rH Φ 4H g CJ Xl 4-1 U W Ti rd SH 0 Xi rH ra Φ ,-, d SH rd 00 rH 4-1 SH rH g ra ■ rl 0 •^ X _-. φ d Φ d rd rd rd Φ • -H ' 3 g 4-1 CQ d 4-1 Φ Ss φ SH 0 TJ ■ rl Φ Q 2 d * -*. SH rd g CM XI rH φ rd Xl -H 0 xl Φ -H Φ SH CQ d ' — •* φ Φ 4-J tn Φ Φ U 4-1 Φ > ft cn O 4-J rH 4J 3s ra 4-J Φ 3 Φ -rl Xl φ -rl d 4-1 Xl 1 ra T! o d -H ■ rl S rd 0 ra CM — >1 SH rd 4J -H 3 4-1 -H ft tn d -H ft 0 rH φ φ Ti φ Xl 4J d -H 4-1 0 d 4-1 3 XI ^ 0 EH ^-^ φ 3 tn d 0 4J Φ xl 4-1 (E,F) Expression of Ptx3 was not detected in the brain at E10.5. Lack of expression in Rathke ' s pouch (RP) , a site of Ptxl and Ptx2 expression (11-14) , confirms the specificity of the hybridisation reaction. (G,H) Higher magnification of the tegmental region expressing Ptx3 at Ell.5. Expression is detected ventrally in the marginal zone (MZ) of the neuroepithelium. (I,J) A coronal section of an E14.5 mouse brain shows the lateral extent of Ptx3 expression in the developing tegmentum (Teg). (K,L) A sagittal section of an E15.5 mouse head shows strong expression of Ptx3 in the ventral tegmentum (vTeg) . In contrast to other hybridisations that were performed with 5' or 3 ' Ptx3 probes, the probe used for this experiment contained the homeodomain and there is weak cross-reactivity with other members of the Ptx family outside the brain, e.g. the pituitary (11-14) (Pit). Mes : mesencephalon; Di : diencephalon; Tel: Telencephalon; Rhom: rhombencephalon; MF : mesencephalic flexure; RP: Rathke ' s pouch; Is: istmus; Pit: pituitary; To: tongue; 01: olfactory epithelium; MZ : marginal zone; VZ : ventricular zone; Str: striatum; vTeg: ventral tegmentum; ct : cartilage of the throat; uLi : upper lip, lLi : lower lip. Figure 5 cDNA nucleic acid sequence of the Ptx3 gene Figure 6 Cloning of Mouse ES-cell genomic DNA Ptx3 cDNA probes spanning the following regions: bp 1-285, 285-799, 799-971, where used in a placque lift/Southern hybridisation protocol to screen a Mouse ES-library. Repetitive screening led to the purification of seven (no 1 to 7 on figure) individual clonses that were purified and analysed for there insert by southern hybridisation using the same probes (see figure) . The restriction analysis together with the southern hybridisation led to the mapping of the first three exon of the ptx3 gene. Note the EcoRI sites in the gnomic clones indicate introns since no EcoRI sites are present in the Ptx3 cDNA. Figure 7 (A) Expression of Ptx3 in the mesDA system of the mouse embryo at stage E 11.5 (B) Expression of Ptx3 in the developing lens. (C) Expression of Ptx3 in sclerotome and in the tongue. Figure 8 Activation of thyroxine hydroxylase promotor by Ptx3

Claims: CLAIMS I. An isolated and/or recombinant nucleic acid or a specific fragment, homologue or derivative thereof, corresponding to a gene with restricted expression in mesencephalic dopaminergic neurons . 2. A nucleic acid according to claim 1 wherein said gene is related to neurological or psychiatric disorders such as Parkinson's disease, tardive dyskinesia, manic depression and schizophrenia . 3. A nucleic acid according to claim 1 or 2 wherein said gene is related to a homeodomain gene, preferably being a bicoid- related homeodomain gene . 4. A nucleic acid according to claim 1, 2 or 3 which is of mammalian origin. 5. A nucleic acid according to claim 4 and hybridising to a nucleic acid with the nucleic acid sequence as listed in figure 5. 6. A nucleic acid according to claim 4 and hybridising to a nucleic acid of mouse origin. 7. A nucleic acid according to claim 4 and hybridising to a nucleic acid of human origin. 8. A method for identifying a gene using a nucleic acid according to any of claims 1 to 7 or using a protein encoded by said nucleic acid. 9. A method according to claim 8 wherein the gene is related to neurological or psychiatric disorders. 10. A method distinguishing between alleles of a gene using a nucleic acid according to any of claims 1 to 7. II. A method for determining expression of a gene using a nucleic acid according to any of claims 1 to 7. 12. A method according to claim 11 wherein the gene is related to neurological or psychiatric disorders. 13. A method according to claim 11 or 12 wherein said expression is determined in neurons. 14. A protein or peptide comprising an amino acid sequence encoded by a nucleic acid according to any of claims 1 to 7. 15. A natural or synthetic antibody directed against a protein or peptide according to claim 14. 16. A method for determining expression of a gene using an antibody according to claim 15. 17. A method according to claim 16 wherein the gene is related to neurological or psychiatric disorders. 18. A method according to claim 16 or 17 wherein said expression is determined in neurons. 19. -A method for detecting a gene product or fragment thereof using a nucleic acid according to any of claims 1 to 7 or an antibody according to claim 15. 20. A method according to claim 19 wherein said gene product or fragment thereof is detected in cerebrospinal fluid. 21. Use of a method according to any one of claims 8 to 13 or 16 to 20 to detect or diagnose a neurological or psychiatric disorder, such as Parkinson's disease, tardive dyskinesia, manic depression and schizophrenia. 22. Use of a method according to any one of claims 8 to 13 or 16 to 20 to test or develop specific medication for the treatment of a neurological or psychiatric disorder, such as Parkinson's disease, tardive dyskinesia, manic depression and schizophrenia . 23. Use of a method according to any one of claims 8 to 13 or 16 to 20 to identify a gene related to neurological disorders. 24. A recombinant expression vector comprising a nucleic acid according to any one of claims 1 to 7. 25. A cell or an animal comprising a vector according to claim 24. 26. A cell or an animal having been deprived of at least a part of a nucleic acid according to any one of claims 1 to 7. 27. A cell or an animal wherein at least a part of a nucleic acid according to any one of claims 1 to 7 has been modified by mutation, deletion and/or insertion. 28. A cell or an animal selected by a method according to any one of claims 8 to 13 or 16 to 20. 29. Use of a method according to any one of claims 8 to 13 or 16 to 20 to select a cell or an animal. 30. Use of a cell or an animal according to any of claims 25 to 28 to test or develop specific medication for the treatment of a neurological or psychiatric disorder, such as Parkinson-' s disease, tardive dyskinesia, manic depression and schizophrenia . 31. Use of a cell according to any of claims 25 to 28 for the treatment of a neurological or psychiatric disorder, such as Parkinson's disease, tardive dyskinesia, manic depression and schizophrenia . 32. A pharmaceutical composition comprising a protein or peptide according to claim 14 or an expression vector according to claim 2 . 33. Use of a pharmaceutical composition according to claim 32 for the treatment of a neurological or psychiatric disorder, such as Parkinson's disease, tardive dyskinesia, manic depression and schizophrenia.

Inventors: BURBACH JOHANNES PETER HENRI, SMIDT MARTEN PIET, BURBACH, JOHANNES, PETER, HENRI, SMIDT, MARTEN, PIET
Applicants: BURBACH JOHANNES PETER HENRI, SMIDT MARTEN PIET, UNIV UTRECHT, BURBACH, JOHANNES, PETER, HENRI, SMIDT, MARTEN, PIET, UNIVERSITEIT UTRECHT
Application year: 1998
Main classification: C12N15
IPC codes: A61K38, C07K14, C12N15