CA2309383A1 - Genes with restricted expression in mesencephalic dopaminergic neurons - Google Patents

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 mesDA 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. Nurr1 is expressed in the mid-brain of mice during embryonic development, but expression is not restricted to this area. Nurr1 activity during embyronic development is thought to be part of a very generalised signaling~mechanism that cells use. Nurr1 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 Nurr1 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 5 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). Hy analysing mesDA
cellular markers in for example a transgenic or mutant cell or animal provided by the invention (for example an animal expr-easing 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 W-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-it 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 tissue, more preferably to a nucleic acid being normally expressed by mesDA neurons of healthy adult individuals but being expressed at altered levels or by less neurons or abnormal temporal and spatial expression pattern in the mesDA
5 system. of a patient with a neurological disorder. A nucleic acid provided by the invention can further be cloned and sequenced and modified by various methods known in the art.
For example, cloning of cDNA/genomic DNA can be achieved by methods available in the art, using for example methods such 10 as a plack-lift protocol, Southern hybridisation, long range PCR -(15;47;48) .
For example, a Ptx3 cDNA clone derived from rat-brain is used as a probe to clone other vertebrate nucleic acid homologous, such as human or mouse cDNA. By using of a plack-lift protocol and Southern hybridisation, cDNA is for example selected from a brain-cDNA library. Also, cDNA sequences and genomic sequences are used to construct primers to be used in a long-range Polymerase Chain Reaction (PCR) for cloning of genomic sequences spanning the coding region of the wanted gene. The invention thus also provides a method for identifying, cloning or sequencing a gene using a nucleic acid according to the invention, especially wherein the nucleic acid is re7_ated to a gene with restricted expression in the mesDA system or the gene is related to neurological disorders.
The invention also provides a method distinguishing between alleles of a gene using a nucleic acid provided by the invention. This allows determining the allelic variation that exists within a gene with restricted expression in mesencephalic dopaminergic neurons or 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.
Such allelic variation can be related to mutations or other genetic modifications that predispose to for example neurological disorders, for example by performing association 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 material and by immunohistochemistry on human, rat and mouse brain tissue without interference with other related homeobox domain proteins.
The invention provides a method for determining expression of a gene using an antibody according to the invention. Ptx3 cDNA is used as an exclusive marker for mesDA
neurons by using parts of the DNA as in-situ hybridisation probes or~by use of the Ptx3 antibodies in immunohistochemical detection methods such as radio- or enzyme-linked immunoassay. Specific antibodies are also used to detect Ptx3 proteins in other material like CSF, by usage of immuno-precipitation. A preferred embodiment of the invention is wherein said gene is related to neurological disorders or wherein said expression is determined in neurons, for example as is achieved by immunohistochemistry.
Specifically associating proteins and peptides other than antibodies can also be used for detection.
The invention also provides a method for detecting a gene product or fragment thereof using a nucleic acid according to the invention, for example by testing for allelic variation that exists within a gene with restricted expression in mesencephalic dopaminergic neurons or 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. Such allelic variation is related to mutations or other genetic modifications that predispose to for example neurological disorders. Such allelic variation can for example be found in by locating mutations, or other variations, in a Ptx3 gene by using methods such as sequencing, restriction fragment length polymorphism, SSCP
analysis and others that are available in the art.
The invention also provides a method for detecting a gene product or fragment thereof or diagnosing a neurological disorder using an antibody or associating protein according to the invention, for example wherein said gene product or 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 nitro-cellulose 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 and when means and methods have been provided, the invention provides an elucidation of the cause of malfunction of the mesDA system and means and methods to study it.
The invention further provides a recombinant expression 5 vector comprising a nucleic acid according the invention.
Such a vector can be a plasmid as for example provided injthe experimental part of the description or a viral vector or a targeting~vector specifically directed at a target locus in a genome of an experimental animal or human. Ptx3 is capable of 10 rescuing Parkinson's disease affected MesDA neurons. DNA
cloning of viral vectors can be used to introduce the Ptx3 cDNA into neuronal cells. For example adenoviral vectors can be used which do not replicate after infection of the cells.
These viruses are amplified in specific cell-lines in vitro.

15 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, herpes virus retrovirus) are modified for expression of for example Ptx3 protein by inserting a cDNA of Ptx3 or Ptx3-derived mutant. Viruses modified accordingly are delivered locally in the substantia nigra of patients for example by micro-cannulation and injection. Yet another example is a viral vector used for ex vivo viral gene transfer in cells. In preparation for grafting into patients, cells are infected in vitro with virus modified to express for example Ptx3 by incubation of virus with cells in culture medium. Cells are then implanted in Parkinson's disease patients.
The invention further provides a cell or an animal comprising a vector according to the invention.
Alternatively, a cell or an animal can be deprived of at least a part of a nucleic acid according to the invention, this is a so-called knock-out animal. Alternatively, the invention provides a cell or an animal wherein at least a part of a nucleic acid according to the invention has been modified by mutation, deletion and/or insertion, this refers especially to a transgenic animal. For example the invention provides Ptx3 transgenic or knock-out animals, by using for example basic cloning techniques and for example DNA
integration by embryonic stem-cell injection (see 56;57;58;59;60;61;62;63). For example, to study the ectopic expression of Ptx3 and mutants of Ptx3 in mammals, transgenic animals are constructed by using specific promoter containing plasmids that drive the expression of the target (Ptx3 in this case) ~in a specific organ or region of cells. The construction of this plasmid is accomplished by cloning Ptx3 cDNA in the sense orientation behind a chosen promoter. This plasmid is introduced into mice by random integration.
Transgenic animals are detected by screening their genomic DNA, by analysis of transgene expression or by staining a transgene marker. Depending on the site of integration in the mouse DNA the Ptx3 expression levels can differ, which can be used to study dosage effects. For example, to create knock-out mice cloned mouse Ptx3 genomic sequences are used to create a construct which lacks for example the first exon. By introducing such a construct by homologous recombination in a mouse embryo a chimeric mouse is constructed. When the knock-out construct is expressed in the germ-line, crossings of these animals lead to heterozygote and homozygote Ptx3-knock-out animals.
The invention further provides a cell or an animal selected by a method according to the invention. Typically, such a cell or animal is characterised by an allelic variation in a gene that is detectable using a nucleic acid and/or a method provided by the invention. The invention thus provides use of a method according the invention to select a cell or an animal. Also, the invention provides use of a cell or an animal according the invention to test or develop specific medication 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. For example, it is possible to find a drug or pharmaceutical composition 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 j 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 Caenorhabditis 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 vitro 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 40°C. The PCR product was used to screen an adult rat hypothalamus library in gtll. Isolated phage DNA was cut with EcoRI and the insert of ~1.2 kb was subcloned into pGEM7Zf(+) and both strands of the insert were sequenced.

Northern analysis. Total RNA extracted from tissues of the adult rat by Rnazol (Biotecx Laboratories Inc., Houston, TX) was fractsonated on a formaldehyde-agarose gels and transferred onto a nylon membrane (Hybond-N, Amersham) by 10 downward capillary-blotting. Blots were hybridised with a 32P=random primed labelled complete Ptx3 cDNA, at 65°C
overnight (15). Autoradiography was performed with a Fujix BAS1000 phosphor-imager (Fuji Photo Film Co., Tokyo, Japan).
15 Cell culture, transfection and gel retardation assays. Murine fibroblast L cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10°s fetal calf serum. L cells were transfected by the calcium phosphate method(16).
Precipitate containing 3 ~g of reporter plasmid, 1 ~Cg of 20 effector plasmid, 1 ~.g of RSV-human growth hormone (hGH) internal control plasmid and 5 ~g of carrier DNA (pSP64 (Promega)) was applied on 1.5*lOscells in 35-mm petri dishes. Medium was changed after 16 hours and cells were harvested 24 hours later and assayed for luciferase activity as described previously (17). Transfections were performed in duplicate at least three times and transfection efficiencies were corrected by measuring hGH in the media using a RIA kit (Immunocorp, Montreal). Nuclear extracts were prepared from 300 000 L cells transfected with 20 ~,g of either a control vector or an expression vector for Ptxl, Ptx3, Otx1 or Otx2 as described( 18). The sequence of the double-stranded oligonucleotides and binding reactions were as previously described ( 12 ) .
In situ hybridisations. Preparation of rat and human brain sections and in situ hybridisations were done as described (19) and sections were exposed on Betamax films (Amersham Life Science, Amersham, UK) for 3 to 7 days, and then subjected to autoradiography under HypercoatTM LM-1 liquid emulsion (Amersham Life Science, Amersham, UK). The cRNA
probes were synthesised from an EcoRI/PstI fragment containing by 1 to 285 and a PstI/BamHI fragment containing by 799-971 of the Ptx3 cDNA. Double labelling was performed with digoxigenine-labelled BalI/EcoRI (bp 915-1137) fragment of the rat TH cDNA20 and the 35S-labelled Ptx3 EcoRI/PstI
fragment. CBAxC57B16 mice were mated and the morning when a vaginal plug was detected was considered E 0.5. Pregnant mice were killed by cervical dislocation, embryos were dissected, fixed overnight at 4°C in 4~ paraformaldehyde and embedded in paraffin. Sections (5 ~.m) were mounted on aminopropylethoxysilane-treated slides and used for in situ hybridisation as described (11,21). Slides were dipped in K.5 autoradiographic emulsion (Ilford), exposed for 25-30 days, developed with Kodak D-19 and counterstained with haematoxylin-eosin.
RESULTS AND DISCUSSION
Cloning of several cDNA clones from rat brain led to the characterisation of a novel cDNA encoding a protein of 302 AA. Comparison of its amino acid sequence to the database (Fig. lA) revealed that it is related to Ptxl and Rieg (hereafter named Ptx2). These two related proteins were implicated in pituitary-specific gene expression and in development of stomodeal structures (11-13), and in the Rieger syndrome, an autosomal-dominant human disorder characterised by craniofacial malformation (14), respectively. Based on homology to Ptxl and Ptx2 (Fig. lA), the protein was called Ptx3. The homeodomains of these three genes are highly conserved, only differing by one or two amino acids, and all display the lysine residue at position 50 of the homeodomain typical for bicoid-related proteins (Fig. lA). The homology also extends into the proximal and extreme C-terminus (67~ homology, Ptx3 vs. Ptxl, Ptx3 vs.
Ptx2). Thus, these three genes constitute a distinct and closely related subfamily within the paired-like class of homeoproteins. Ptx3 is modular of primary structure. Within the Ptx3 primary structure several functional and structural domains can be addressed (Figure 11). 1) the Homeodomain: of the bicoid like and paired like class of homeodomains. The bicoid like characteristic is based on residue 9 of the third helix of the homeodomain which is in that case a lysine (K).
2) The conserved C-terminal peptide. 3) The far C-terminus is an attenuating domain which alters the activity of the transactivation domain. Ptx3 mutants lacking the last 68 amino acids (Ptx3 as 1-234) or the amino acid sequence (aa 280-292) are more powerful transactivators than the complete Ptx3 protein (aa 1-302).
Since Ptx3 is a new member of the Ptx-subfamily we compared its binding and trans-activation properties to those of Ptxl and of the related bicoid-like homeodomain proteins Otx-1 and -225. In DNA-binding experiments all four homeodomain proteins bound the Ptxl-binding site of the pro-opiomelanocortin (POMC) gene (Fig. 1B). In contrast, both Ptx3 and Ptxl exhibited stronger transactivating potential than Otx-1 and -2 on a reporter containing three Ptxl binding sites (Fig. 1C). This activity was completely abolished by a mutation that prevents DNA-binding (Fig. 1B) as previously shown for Ptxl(11). Thus, Ptx3 can function as an activator of transcription upon direct interaction with target genes.
Furthermore, Ptx3 can activate the tyrosine hydroxylase promoter in vitro. Ptx3 is able to enhance the transcription of the -750 /+64 Tyrosine Hydroxylase promoter in vitro in cell-lines using Luciferase as a promoter. Deletion of the promoter beyond -500 reduces the transactivation (Figure 8).
In order to develop an hypothesis on the function of Ptx3, its expression was analysed in various tissues, including the pituitary gland which is an important site of expression of Ptxl and Ptx2 (11-14). Northern-blot analysis indicated that Ptx3 is expressed in rat E19 embryo head.
However, it could not be detected in dissected adult rat brain regions or in a set of peripheral organs (Fig. 2). Ptx3 is not expressed in the anterior pituitary or in a number.of cell lines of pituitary origin (not shown), in contrast to Ptxl and Ptx2. Thus, Ptx3 appears to have a very different expression pattern compared to the other subfamily members.
Since Ptx3 was not detected in dissected brain regians on Northern blots, despite of its cloning from brain RNA, in situ hybridisation was used to determine if Ptx3 was expressed in a restricted manner in the brain. At the macroscopic level, hybridisation of Ptx3 was confined to the substantia nigra pars compacta (SNc; A9) and the ventral tegmental area (VTA; A10) (Fig. 3A), together harbouring the mesDA system(26), but was not seen in rostral regions having dopaminergic neurons, nor in the adrenal gland (Fig. 2). In order to determine if Ptx3 is indeed expressed in dopaminergic neurons we performed double in-situ hybridisation with Ptx3 and tyrosine hydroxylase (TH) cRNA
probes. Ptx3 expression completely overlapped with TH-positive cells, demonstrating that Ptx3 is expressed in dopaminergic neurons of the mesDA system. Ptx3 is specifically expressed in time and space. First, in the MesDA
system of the rat at stage E 12.5 and in the mouse at stage E
11.5 (Figure 7A). This expression continues at high levels until the end of life. Expression of Ptx3 is also in the eye of the developing rat and mouse embryo where expression of Ptx3 is found, especially in the developing lens (Figure 7B).
Third, moderate Ptx3 expression is seen in sclerotome and in the tong at stage E14.5 ( Figure 7C) in the rat, this expression disappears at birth. Thus, apart from being involved in mesDA development, Ptx3 may play a role in ocular and cartilage/muscle development as well. Exclusive expression during embyronic development of a homeobox gene WO 99/24572 PCTlNL98/00652 related to Pitx/Rieg family was found recently in the eye of the mouse (64).
In situ hybridisation experiments on the human substantia nigra showed co-localisation of the human counterpart of Ptx3 with pigmented cells which represent the mesDA neurons. Furthermore, in-situ analysis of the substantia nigra of Parkinson patients revealed a reduced density of Ptx3-expressing neurons as compared to normal controls. Thus, the loss of expression correlates with loss of the mesDA neurons, both in animal models and human disease. Taken together, the results clearly indicate that mesDA neurons express Ptx3 both in rat and man.
As homeodomain proteins are usually involved in pattern formation, the close association of Ptx3 expression with an intact mesDA system shows that Ptx3 is involved in development and/or maintenance of this subset of dopaminergic neurons. Sectioned mouse embryos from E8.5 to E16.5 were examined by in situ hybridisation in order to correlate Ptx3 expression with development of mesDA neurons. No signal above background was detected in the head at E8.5 (not shown) and at E10.5 (Fig. 4F). At E11.5, a small layer at the ventral surface of the mesencephalic flexure expressed Ptx3 (Fig.
4B). This group of about 50 cells corresponds to the first TH-expressing cells in the developing rodent brain (3-5,26).
At later stages, the expression remained restricted to the mesDA system (Fig 4D,J,L) and this association is conserved in adult rat brain (Fig. 3). Higher magnifications show that Ptx3-positive cells are restricted to the marginal layer of the mesencephalic tegmentum (Fig. 4H); a coronal section at E14.5 reveals that the signal is restricted to the central region of the ventral tegmentum (Fig 4J). Apart from the mesDA system, Ptx3 expression in the sclerotome and its cartilaginous derivatives, in the eye and in the tongue was detected at this stage (Fig 4L). The developmental pattern in brain shows that Ptx3 expression coincides spatially and temporal with the appearance of the developing mesDA system.

While the combinatorial action of many transcription and growth factors is likely involved in brain patterning (29,30), the present work suggests that single homeodomain transcription factors may determine single neuronal cell 5 identity. We have shown a restricted expression of Ptx3 in one neuronal lineage, the mesDA neurons. The only other example of such restricted neuronal expression is for a nematode gene, Unc-30, the closest invertebrate homologue of the Ptx-family. This gene is specifically required for the 10 differentiation of the inhibitory GABA-ergic type D motor neurons of this nematode (31). The mesDA system is functionally heterogeneous and anatomically differentiated by different fields of axonal afferents (26,32). Ptx3 is expressed in all these fields and thus, its developmental 15 association with the mesDA system shows that Ptx3 plays a part in the development of the dopaminergic cell-type rather than in axonal pathfinding or establishment of specific connectivity. Other factors involved in mesDA neuron development include the signalling molecule sonic hedgehog 20 (33) and the orphan nuclear receptor Nurrl (34). Nurrl is expressed from E 10.5 in the mouse midbrain but not restricted to this area. Nonetheless, Nurrl knock-out mice specifically failed to develop mesDA neurons (35). These data suggest that Ptx3 and Nurrl form a regulatory cascade for 25 development of the mesDA system in which Nurrl acts as an upstream activator. Since, Ptx3 in contrast to Nurrl, shows restricted expression in the mesDA neuronal system, it activates a program for mesDA-specific gene expression and differentiation.
The degeneration of dopaminergic neurons within the mesDA system is the direct cause of Parkinson's disease (7-10), while other extrapyramidal motor disorders like tardive dyskinesia (36-38) are also associated with this system.
Furthermore, the mesDA system has been implicated in affective disorders like manic depression and schizophrenia (6,39), and in behavioural reinforcement and drug addiction (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.
Furthermore, the function of Ptx3 can be exploited for manipulation of dopaminergic cells in vitro in preparation of tissue grafting, an experimental therapy for Parkinson patients (41-43).

1. Lumsden, A and Krumlauf, R. (1996) Science 274, 1109-1115.
2. Tanabe, Y. and Jessell, T.M. ibid., 1123.
3. Specht, L.A., Pickel, V.M., Joh, T.H. & Reis, D. (1981) J.
Comp. Neurol. 199, 233-253.
4. Voorn, P., Kalsbeek, A., Jorritsma-Byham, J. &
Groenewegen, H.J. (1988) Neuroscience 25, 857-887.
5. Solberg, Y., Silverman, W. & Pollack, Y. (1993) Dev. Brain Res. 73, 91-97.
6. Grace, A.A., Bunney, B.S., Moore, H. & Todd, C.L. (1997) Trends Neurosci. 20, 31-32.
7. Jellinger, K. (1973) Cesk. Pathol. 9, 1-13.
8. Forno, L.S. (1992) Ann. N Y Acad. Sci. 648, 6-16.
9. Golbe, L.I. (1993) Rev. Neurosci. 4, 1-16.
10. Hirsch, E.C. (1994) Mol. Neurobiol. 9, 135-142.
11. Lamonerie, T., Tremblay, J.J., Lanctot, C., Therrien, M., Gauthier, Y. & Drouin, J (1996) Genes Dev. 10, 1284-1295.
12. Lanctot, C., Lamolet, B. and Drouin, J. (1997) Development 124, 2807-2817.
13. Szeto, D.P. , Ryan, A.K., O'Connell, S.M. & Rosenfeld, M.G. (1996) Proc. Natl. Acad. Sci. USA 93, 7706-7710.
14. Semina, E.V., Reiter, R., Leysens, N.J., Alward, W.L.M., Small, K.W., Datson, N.A., Siegel-Bartelt, J., Bierke-Nelson, D., Bitoun, P., Zabel, B.U., Carey, J.C., & Murray, J. (1996) Nature Gen. 14, 392-399.
15. Church, G.M. & Gilbert, W. (1984) Proc. Natl. Acad. Sci.
USA 81, 1991-1995.
16. Chen, C. & Okayama, H. (1987) MoI Cell. Biol. 7, 2745-2752.
17. Therrien, M. & Drouin, J. (1991) Mol. cell. Biol. 11, 3492-3503.
18. Schreiber, E., Matthias, P., Miiller, M.M. & Schaffner, W.
(1989) Nucleic Acids Res. 11, 6419.
19. Lopes da Silva, S., Horssen, A.M. van, Chang, C. and Burbach, J.P.H. (1995) Endocrinology 136, 2276-2283.

20. Grima, B., Lamouroux, A., Blanot, F., Biguet, N.F. &
Mallet, J. (1985) Proc. Natl. Acad. Sci.USA 82, 617-621.
21. Wilkinson, D.G. (1992) in In situ hybridisation a practical approuch (ed. D.G. Wilkinson) IRL press, New York, NY.
22. Van Ree, J.M. & Wolterink, G. (1981) Eur. J. Pharmacor.
72, 107-111.
23. Biirglin, T.T. (1993) in A Guidebook for Homeobook Genes (ed. D. Duboule; Oxford university press, Oxford, UK), pp 25-71.
24.-LaCasse, E.C. & Lefebvre, Y.A. (1995) Nucleic Acids Res.
23, 1647-1656.
25. Simeone, A., Acampora, D., Gulisano, M., Stornaiuolo, A.
& Boncinelli, E. (1992) Nature 358, 687-690.
26. Bjorkland, L.A. & Lindvalt, O. (1984) in Handbook of Chemical Neuroanatomie, Elsevier science publischer, ed.
Bjorkland, L.A. & Hokfelt, T., 2, pp 55-122 27. Ungerstedt, U., Ljungberg, T. & Steg, G. (1974) Adv.
Neurol. 5, 421-426.
28. Hudson, J.L., Van Horne, C.G., Stromberg, I., Brock, S., Clayton, J., Masserano, J., Hoffer, B.J. & Gerhardt, G.A.
(1993) Brain Res. 626, 167-174.

29. Rubenstein, J.L.R., Salvador, M., Shimamura, K. &
Puelles, L. (1994) Science 266, 578-580.

30. Figdor, M.C. & Stern, C.D. (1993) Nature 363, 630-634.

31. Jin, Y., Hoskins, R. & Horvitz, R. (1994) Nature 372, 780-783.

32. Fallon, J.H., & Loughlin, S.E. (1995) in The rat nervous system second edition, ed. G. Paxinos, school of Psychology, Syndey, Australia, Acad. Press., 215-237.

33. Hynes, M, Porter, J.A., Chiang, C., Chang, D., Tessier-Lavigne, M., Beachy, P.A. & Rosenthal, A. (1995) Neuron 15, 35-44.

34. Zetterstrom, R.H., Williams, R., Perlmann, T. & Olson, L.
(1996) Mol. Brain Res. 41, 111-120.

35. Zetterstrom, R.H., Solomin, L., Jansson, L, Hoffer, B.J, Olson, L. & Perlmann, T. (1997) Science 276, 248-250.

36. Klawans, H.L., Goetz, C.G. & Perlik, S. (1980) Am. J.
Psychiatry 137, 900-908.

37. Goetz, C.G. & Klawans, H.L. (1984) Neurol. Clin. 2, 605-614.

38. Verghese, C., De Leon, J. & Simpson, G.M. (1994) Am. J.
Psychiatry 151, 1716-1717.

39. Crow, T.J., Johnstone, E.C., Longden, A. & Owen, F.
(1978) Adv. Biochem. Psychopharmacol. 19, 301-309.

40.~White, F.J. (1996) Annu. Rev. Neurosci. 19, 405-436.

41. Perlow, M.J. (1987) Neurosurgery 20, 335-342.

42. Date, I., Miyoshi, Y., Ono, T., Imaoka, T., Furuta, T., Asari, S., Ohmoto, T. & Iwata H. (1996) Cell Transplant 5, S17-519.

43. Kordower, J.H., Rosenstein, J.M., Collier, T.J., Burke, M.A., Chen, E.Y., Li, J.M., Martel, L., Levey, A.E., Mufson, E.J., Freeman, T.B. & Olanow, C.W. (1996) J. Comp. Neurol.
370, 203-230.

44. Wong, C. & Naumovski, L. (1997), Anal. Biochem. 252, 33-39.; Kato, M., Sasaki, T., Ohya, T., Nakanishi, H., Nishi.oka, H., Imamura, M. & Takai, Y. (1996), J. Biol. Chem. 271, 31775-31778.;

45. White, M.A. (1996), Proc. Natl. Acad. Sci. USA 93, 10001-10003.;

46. Luban, J. & Goff, S.P. (1995), Curr. Opin. Biotechnol. 6, 59-64;

47. Ausubel, F.M., Brent, T., Kingston, R.E., More, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (1994) Current Protocols in Molecular Biology, Massachusetts general hospital, Harvard Medical School).

48. Gordadze, A.V., Benes, H. (1996), Biotechniques 21(6), 1062-1066. ) .

49. Sanger, F., Nicklen, S. & Coulson, A.R. (1977), Proc.
Natl. Acad. Sci USA 74, 5463-5467.

50. Date, Y., Nakazato, M., Kangawa, K., Shirieda, K., Fujimoto, T. & Matsukura, S. (1997) J. Neurol. Sci. 150, 143-148.;

51. Petry, F., Berkel, A.I. & Loos, M. Hum. Genet. 100, 51-5 56.

52. Pena, S.D., Prado, V.F. & Epplen, J.T. (1995) J. Mol:
Med. 73, 555-564.;

53. Humphries, S.E., Gudnason, V., Whittall, R. & Day. I.N.
(1997) Clin. Chem. 43, 427-435).
10 54. Wang, X., Ruffolo, R.R. Jr & Feuerstein, G.Z. (1996), Trends Pharmacol. Sci. 17, 276-279.;
55. Wang, X., Ruffolo, R.R. Jr & Feuerstein, G.Z. (1996), Trends Pharmacol. Sci. 17, 276-279.;
56. Wilder, P.J . & Rizzino, A., (1993} Cytotechnology 11, 79-15 99.
57. Shibata, H., Kanamaru, R. & Noda, T. {1997) Gan. To.
Kagaku. Ryoho. 24, 460-465.
58. Pomeroy, K.O. (1991} Genet Anal. Tech. Appl. 8, 95-101.
59. Zakany, J., Tuggle, C.K. & Nguyen-Huu, C.M. (1990), J.
20 Physiol. 84, 21-26.
60. 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.;
25 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.).
30 64. Semina, E.V., Reiter, R.S. and Murray, J.C. (1997) Hum.Mol.Gen. 6, 2109-2116.

FIGURE LEGENDS
Figure 1. Structure and properties of PTX3.
(A) Primary structure of Ptx3 deduced from the open reading frame of the Ptx3 cDNA and alignment to Ptxl(11-13) and Ptx2 (RGS)(14). The homeodomain is underlined with the bicoid-type specific lysine at position 9 of the third helix double(23).
A consensus nuclear localisation signal is shown in bold italics(24). The novel conserved C-terminal domain(14) is typed in bold-face.
(B) Binding of Ptx3 to the CE3 element of the rat POMC
promoter in gel shift analysis. Ptx3 binds to the CE3 element of the rat POMC promoter as was found for the related factors Ptxl and Otxl and Otx2. Competition experiments using mutant recognition sites(11) within (M1) - and outside the bicoid core (M3) of the CE3 element (200 fold molar excess) shows that the ineffective mutant M3 and the wild-type compete for Ptx3 binding whereas the effective mutant M1 cannot.
(C) Transactivational properties of ptx3 in transient transfection assays. The activity of Ptx3 on an artificial promoter construct based on three copies of the POMC-CE3 element are comparable to that of Ptx111, whereas Otxl and Otx2 are clearly less active. When using the M1 mutated binding site the activity is lost.
Figure 2. Northern analysis of Ptx3 expression in the adult rat brain and peripheral organs.
Blots containing 20 ~.g of total RNA of dissected rat brain regions and selected peripheral organs were hybridised with a full length Ptx3 cDNA probe. GapDh was used as a control. The positions of 18S and 28S ribosomal RNAs are indicated by arrows.

Figure 3. Expression of Ptx3 in the mesencephalic dopaminergic system of rat and man.
(A) The expression pattern of Ptx3 in rat brain starting anterior at the substantia nigra compacta (SNc) to posterior at the ventral tegmental area (VTA). The size bar in the upper panel represents a distance of 1 mm. The rostral-caudal positions of the first and last panels are indicated as mm relative to bregma in the upper right corner.
(B) Double in-situ hybridisations using a DIG-labelled tyrosine hydroxylase (TH) cRNA probe and a 35S-labelled Ptx3 cRNA probe shows a similar hybridisation pattern for both probes in the tegmentum of the rat brain (upper two panels).
Microscopic examination (middle panels) shows TH positive cells in blue and Ptx3 labelled by silver grains (left panel:
darkfield image, right panel: higher magnification, bright field image). A complete overlap is found.
(C) Ptx3 expression in the human substantia nigra of two healthy controls (control land 2) and two Parkinson patients (Patient 1 and 2). The sections of the patients show a lower density of Ptx3 expressing cells which correlates with the loss of dopaminergic neurons within this region. The lower panel represents a dark-field image showing Ptx3 expression (silver grains) co-localising with typical brown pigmented dopaminergic neurons of the human substantia nigra. Material was obtained from the Netherlands Brain Bank. Patient material was diagnosed clinically and pathologically.
Figure 4. In situ hybridisation of Ptx3 in embryonic mouse brain sections between E10.5 and E15.5.
Micrographs of bright field and darkfield images of the same sections are shown. (A,B) At E11.5, expression is seen in a layer of postmitotic cells (arrow) in the mesencephalon (Mes) lining the mesencephalic flexure (MF}. (C,D) At E12.5 the layer of expressing cells has thickened as part of the developing tegmentum (Teg). Extraneural expression is seen in the tongue (To), but not in the developing pituitary (Pit).

(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 E11.5. 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; Ol: 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: by 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 (33)

1. 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.

11. 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 24 or an expression vector according to claim 24.

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.