WO2003102587A1

WO2003102587A1 - Disc1 partners

Info

Publication number: WO2003102587A1
Application number: PCT/GB2003/002396
Authority: WO
Inventors: Kirsty Millar; David John Porteous; Walter John Muir; Douglas Blackwood
Original assignee: University Court Of The University Of Edinburgh
Priority date: 2002-06-01
Filing date: 2003-06-02
Publication date: 2003-12-11
Also published as: AU2003273535A1; GB2405472B; GB2405472A; GB0426454D0; GB0212856D0

Abstract

The present invention relates to a method of screening for agents which disrupt or enhance the binding of DISC1 partners to DISC1 polypeptide. The present invention also relates to a method of diagnosing and treating psychiatric disorders, in particular, schizophrenia and other psychotic and mood disorders.

Description

D1SC1 PARTNERS The present invention relates to a method of screening for agents which disrupt or enhance the binding of DISC1 partners to DISC1 polypeptide. The present invention also relates to a method of diagnosing and treating psychiatric disorders, in particular, schizophrenia and other psychotic and mood disorders.

Schizophrenia and other psychotic and mood disorders are common and debilitating psychiatric disorders. Despite a wealth of information on the epidemiology, neuroanatomy and pharmacology of the illness, it is uncertain what molecular pathways are involved and how impairments in these affect brain development and neuronal function. Despite an estimated heritability of 60-80%, very little is known about the number or identity of genes involved in these psychoses.

In a large Scottish family, a balanced translocation (1 ;11)(q42;q14) co-segregates with major psychiatric disorders (schizophrenia, schizoaffective disorder and other major affective disorders) with a maximum LOD score of 7.1 (St Clair et al, Lancet 336: 13-16, Blackwood et al, Am J Hum Genet 69: 428-433, 2001). Individuals carrying the translocation are therefore predisposed to developing major psychiatric illness. The translocation directly disrupts DISC1 (Disrupted In Schizophrenia 1) and consequently this gene is suspected to be a schizophrenia susceptibility gene (Millar et al, Hum Mol Genet 9: 1415-1423 2000). Furthermore, DISC1 is located within a region of the human genome identified as a schizophrenia susceptibility locus (LOD=3.21 , Ekeiund et al, Hum Mol Genet 10: 1611-1617, 2001). It is proposed that studying the function of DISC1 will elucidate the mechanisms causing susceptibility to severe psychiatric illnesses in this family and others, and furthermore, may point towards alternative, but related, molecular defects in psychiatric patients where DISC1 is not at fault.

The present inventors have identified five proteins which interact with DISC1 polypeptide. It is postulated that normal in vivo binding to one or more of these proteins by DISC1 may be disrupted in individuals who are susceptible to or have a psychiatric disorder.

Therefore, an object of the present invention is to provide a method of screening for agents which disrupt or enhance the binding of DISC1 partners to the DISC1 polypeptide. A further object of the present invention is to provide a method of treating psychiatric disorders, in particular, schizophrenia and other psychotic and mood disorders, such as bipolar affective disorder.

In a first aspect, the present invention provides a method of screening for a candidate agent which modulates the interaction between a DISC1 partner polypeptide and DISC1 polypeptide, said method comprising:

(a) forming a mixture comprising the candidate agent, a DISC1 partner polypeptide, selected from the group consisting of ATF4, KIAA1167, KIAA0380, AKAP450 and mNUDE/NUDE1 , or splice variants, mutant, fragments, or orthologues thereof, and DISC1 polypeptide; (b) subjecting the mixture to conditions suitable for allowing a native DISC1 partner polypeptide and a native DISC1 polypeptide to bind to one another in the absence of any agent; and

(c) detecting any modulation in the binding of said DISC1 partner to said DISC1 polypeptide. The term "DISC1 partner" refers to any one of the polynucleotide sequences and/or polypeptides disclosed herein, or splice variants, fragments, or orthologues thereof which interact with the DISC1 polypeptide. In particular, the term is intended to include, but not be limited to, the polynucleotide and polypeptide sequences, as illustrated in Figures 1A (ATF4), 2A (KIAA1167), 3A, 3C (KIAA0380), 4A, 4C, 4E (AKAP450), 5A, 5C and 5E (mNUDE), and Figures 1B (ATF4), 2B (KIAA1167), 3B, 3D (KIAA0380), 4B, 4D, 4F (AKAP450), 5B, 5D and 5F (mNUDE), respectively. Hereinafter mNUDE will be referred to as NUDE1 , as this is the name most commonly given to the gene/protein. It is understood that any mutant form of the sequence may comprise a deletion, substitution, invasion or translocation of sequence with respect to the sequences shown. The term "DISC1 polypeptide" refers to the protein, or fragments thereof, described in

PCT published application WO 01/40301 (referred to therein as "DIS1"). The protein disclosed therein was only recently identified as being involved in schizophrenia, other psychotic disorders and mood disorder. The polypeptides of the present invention have been identified through their binding with the DISC1 polypeptide. The term "polynucleotide sequence" generally refers to DNA but is understood to be non-limiting and may include RNA, cDNA, etc. The term may also be used interchangeably herein with the terms "nucleotide", "gene" and "genetic sequence".

A polynucleotide fragment is understood to include, but is not limited to, a polynucleotide sequence encoding one or more of the polypeptide regions or domains of the DISC1 partners which interact with the DISC1 polypeptide, or a fragment thereof. It may, for example, be from 10 nucleotides long, selected from any area of the coding region of the polynucleotide sequence, up to the full length of the sequence. A polypeptide fragment is understood to include, but is not limited to, a sequence of amino acids from, for example, 5 amino acids long up to the full length of the polypeptide. It also encompasses one or more of the polypeptide regions or domains of the DISC1 partners which interact with the DISC1 polypeptide.

For the purposes of the present invention, the terms "modulates" or "modulation" are regarded as an increase or decrease in the binding affinity between the DISC1 partner polypeptide and the DISC1 polypeptide.

Agents which affect the binding of said DISC1 partner polypeptides with said DISC1 polypeptide may include, but are not limited to, small molecules and/or peptides and larger molecules such as proteins, for example, antibodies. Furthermore, these agents may be existing compounds or may be newly synthesised, or may be drugs which have been used to treat disease conditions other than those described herein.

Methods of detecting an interaction between the DISC1 partner polypeptide and DISC1 polypeptide include, but are not limited to, FRET assays (see Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations. Sekar RB, Periasamy A. J Cell Biol 2003 160(5) :629-33), gel retardation assays, co-precipitation assays, double fluorescence/cell staining protocols, GST pull-down and other affinity purification methods, and yeast two-hybrid system. All such techniques are known to those skilled in the art and are generally described in Molecular Cloning (3-volume set), Joe Sambrook and David Russell (2000) Cold Spring Harbor Laboratory Press) or Current Protocols in Molecular Biology, edited by Frederick M. Ausubel, Roger Brent, Robert E. Kingston, David D. Moore, J.G. Seidman, John A. Smith & Kevin Struhl. Massachusetts General Hospital and Harvard Medical School. Other molecular biological techniques mentioned throughout this specification may be also carried out as described in either of these references.

The assay according to the present invention may be easily adapted to be used for high throughput screening of libraries of compounds, such as synthetic, natural or combinatorial compound libraries.

Any one of the proteins of the present invention and the DISC1 polypeptide may be used in isolation i.e. in vitro. Alternatively, the polypeptide of the present invention may be expressed in a cell line or transgenic animal as described herein. Furthermore, said DISC1 partner polypeptide and the DISC1 polypeptide used in the assay of the present invention may be native, normal polypeptides i.e. unmutated, or may each independently be mutated i.e the polynucleotide and/or polypeptide sequences may be altered compared with the native version of the polypeptides or protein fragments. The use of mutated sequences may help to identify agents which restore the wild-type functional activity of either a mutated DISC1 polypeptide or a mutated form of any one of the DISC1 partner polypeptides disclosed herein.

As discussed above, the sequences of the present invention are intended to encompass splice variants of the genes. For example, the KIAA0380 gene structure has been annotated from the human genome sequence and consists of 40 exons extending across 110 kb (human genome browser, http://genome.ucsc.edu/index.html). Further investigation of KIAA0380 transcripts using the human genome browser reveals that this gene is alternatively spliced. Ah additional exon is included in at least seven transcripts (designated exon 34). This exon is flanked by consensus splice sites and consists of 120 nucleotides. Inclusion of this exon will therefore maintain the open reading frame. Exon 34 encodes the amino acids RICEVYSRNPASLLEEQIEGARRRVTQLQLKIQQETGGSV. This exon is not included in the only orthologous rat transcript currently available (GenEMBL accession number AF225961). However, comparisons with mouse genomic and transcribed sequence (GenBank accession numbers AZ729400, AI427281 and W47871) indicate that this exon is highly conserved (97%) and utilised in transcripts, further strengthening the evidence that this is a true exon. The position of inclusion of exon 34 is 1327 in sequence KIAA0380. ln addition, the AKAP450 gene structure has been annotated from the human genome sequence and consists of 51 exons extending across 200 kb (human genome browser, http://genome.ucsc.edu/index.html). Further investigation of AKAP450 transcripts using the human genome browser reveals that this gene is alternatively spliced as follows: 1) One transcript (GenEMBL accession number AL117418) utilises an alternative exon 1, designated 1b. This exon possesses a donor splice site conforming to the consensus sequence, and introduces amino . acids MDSYSTYLATVKVSGSWLEEQDEDIYEAESRV

PLPHPFPLCEHLDENNSVIVNTSIFHFIHKRNGHILKLISKISLPTPPYS into the N-terminus of the polypeptide, replacing those amino acids encoded by exons 1-23. The coding sequence of this exon is extremely highly conserved, maintaining 75% identity upon translation in Tetraodon nigroviridis and Takifugu rubripes (GenEMBL accession numbers

AC113582 and AC098643, respectively). Therefore, although to date utilisation of this exon has been detected in only one human transcript, there is strong evidence that this is a true exon. Utilisation of exon 1 b is predicted to result in production of an N-terminally truncated polypeptide of 2122 amino acids, compared to 3908 amino acids in the full-length polypeptide. The site of exon 1 b inclusion is 5826 in sequence AKAP450.

2) Exon 28 contains an internal consensus splice donor site utilised in two transcripts

(GenEMBL accession numbers AB019691 , AF091711). Use of this splice site results in loss of 24 nucleotides from the transcript (and eight amino acids, VSADTFQK), while maintaining the open reading frame. This exon has been designated exon 28a. This splice site is located at position 6708 in sequence AKAP450.

Moreover, the NUDE1 gene structure has been annotated from the human genome sequence and consists of 9 exons extending across 75 kb (human genome browser, http://genome.ucsc.edu/index.html). This gene is antisense to smooth muscle myosin heavy chain 11 (MYH11) and exon 9 of NUDE1 overlaps exons of MYH11. However, further investigation of NUDE1 transcripts using the human genome browser revealed that NUDE1 is alternatively spliced. Three terminal exons are commonly used, as indicated in each case by several transcripts. In addition to the previously identified exon located within MYH11, there is an alternative final exon that possesses two splice acceptor sites, predicted to generate two alternative C-termini in the NUDE1 polypeptide. Consequently it is likely that three isoforms of NUDE1 exist, each differing in their C-terminal amino acids. Inclusion of exon 9a results in a transcript encoding the amino acids REN at the C-terminus, inclusion of exon 9b results in a transcript encoding the amino acids LGKRLEFGKPPSHMSSSPLPSAQGWKMLL, and inclusion of exon 9c results in a transcript encoding the amino acids LDTSCRWLSKSTTRSSSSC. The C-terminus encoded by exon 9b is highly similar to that of the rat and mouse NUDE1 orthologues. The three isoforms have been designated NUDEIa, NUDEI b and NUDEIc. The position of alternative splicing is nucleotide number 1062 in sequences NUDEIa, NUDEI b and NUDEIc. The invention still further provides an assay as hereinbefore described comprising polypeptides encoded by polynucleotide sequences which are similar to the disclosed polynucleotide sequences. By "similar" it is meant a sequence which is capable of hybridising to a sequence which is complementary to the disclosed polynucleotide sequences. When a similar sequence and disclosed sequence are each double stranded, the nucleic acid constituting the similar sequence preferably has a T_m within 20^CC of that of the disclosed sequence.

In the case that the disclosed inventive sequences are mixed together and denatured simultaneously, the T_m values of the sequences are preferably within 10°C of each other. More preferably, hybridisation may be performed under stringent conditions, with either the similar or disclosed DNA preferably being supported. Thus, for example, either the denatured similar or disclosed sequence is preferably first bound to a support and hybridisation may be affected for a specified period of time at a temperature of between 50 and 70°C in double strength SCC (2 x NaCl 17.5g/l and sodium citrate (SC) at 8.8g/l) buffered saline containing 0.1% sodium dodecyl sulphate (SDS) followed by rinsing of the support at the same temperature but with a buffer having a reduced SSC concentration. Depending on the degree of stringency required, and thus the degree of similarity of the sequences, such reduced concentration buffers are typically single strength SSC containing 0.1% SDS, half strength SSC containing 0.1% SDS and one tenth strength SSC containing 0.1% SDS. Sequences having the greatest degree of similarity are those the hybridisation of which is least affected by washing in buffers of reduced concentration. It is most preferred that the similar and disclosed sequences are so similar that the hybridisation between them is substantially unaffected by washing or incubation at high stringency, for example, in one tenth strength sodium citrate buffer containing 0.1 % SDS.

Therefore, the invention still further provides an assay as hereinbefore described comprising a polypeptide encoded by a polynucleotide sequence which is complementary to the one which hybridises under stringent conditions with the polynucleotide sequences disclosed herein. The polynucleotide sequences are between 70% and 99% similar, for example, 70%, 80%, 90%, 95% or 98% similar, with the disclosed sequences.

As is well known in the art, the degeneracy of the genetic code promotes substitution of bases in a codon resulting in a different codon which is still capable of coding for the same amino acid, for example, a gene codon for amino acid glutamic acid is both GAT and GAA. Consequently, it is clear that for the expression of polypeptides with the amino acid sequences shown in Figures 1 B, 2B, 3B, 3D, 4B, 4D, 4F, 5B, 5D and 5F, or fragments or orthologues thereof, use can be made of derivative nucleic acid sequences with such an alternative codon composition different from the nucleic acid sequences showing in Figures 1A, 2A, 3A, 3C, 4A, 4C, 4E, 5A, 5C and 5E.

For recombinant production of the enzyme in a host organism, the disclosed polynucleotide sequences encoding any one of the DISC1 partner polypeptides may be inserted into an expression cassette to form a DNA construct designed for a chosen host and introduced into the host where it is recombinantly produced. The choice of specific regulatory sequences such as promoter, signal sequence, 5' and 3' untranslated sequences, enhancer and terminator appropriate for the chosen host is within the level of skill of the routine worker in the art. The resultant molecule, containing the individual elements linked in a proper reading frame, may be introduced into the chosen cell using techniques well known to those in the art, such as calcium phosphate precipitation, electroporation, biolistic introduction, lipoplex introduction, polyamine introduction, virus introduction, etc. Suitable expression cassettes and vectors and methods for recombinant production of proteins are well known for host organisms such as E. coli (see eg. Studier and Moffatt, J. Mol. Biol. 189: 113 (1986); Brosius, DNA 8: 759 (1989)), yeast (see eg. Schneider and Guarente, Meth. Enzymol 194: 373 (1991)) and insect cells (see eg. Luckow and Summers, Bio/Technol. 6: 47 (1988)) and mammalian cell (tissue culture or gene therapy) by transfection (Schenborn ET, Goiffon V. Methods Mol Bio. 2000; 130: 135-45, Schenbom ET, Oler J. Methods Mol Biol. 2000; 130: 155-64), electroporation (Heiser WC. Methods Mol Biol. 2000; 130: 117-34) or recombinant viruses (Walther W, Stein U ; Drugs 2000 Aug; 60(2) : 249- 71).

Therefore, the invention further provides an expression cassette comprising a promoter operably linked to a polynucleotide sequence as disclosed herein encoding a DISC1 partner polypeptide, or functionally active variant thereof.

In a further aspect, the present invention also provides a cell line comprising a polynucleotide fragment comprising a polynucleotide sequence of any one of the DISC1 partners disclosed herein, or a fragment, derivative, or orthologue thereof. The cell line may, for example, be a mammalian cell line and, in particular, may be a human cell line. The cell line (which may be transformed) according to the present invention may be used to screen for and evaluate potential agents which may be effective for reducing or substantially eliminating the symptoms of schizophrenia, other psychotic disorders and mood disorders as defined hereinbelow.

In a yet further aspect, the present invention provides a polynucleotide sequence comprising a transcriptional regulatory sequence, a sequence under the transcriptional control thereof which includes an RNA sequence characterised in that the RNA sequence is anti-sense to a mRNA with a polynucleotide sequence of any one of the DISC1 partners disclosed herein. The polynucleotide sequence encoding the anti-sense molecule can be of any length provided that the anti-sense RNA molecule transcribable therefrom is sufficiently long so as to form a complex with a sense mRNA molecule with a polynucleotide sequence of any one of the DISC1 partners disclosed herein. Thus, without the intention of being bound by theory, it is thought that the anti-sense RNA molecule complexes with the mRNA coding for the polypeptide and prevents or substantially inhibits the synthesis of a functional DISC1 partner polypeptide. As a consequence of the interference by the anti-sense RNA, polypeptide levels of the DISC1 partner polypeptide are decreased or substantially eliminated. The polynucleotide sequence encoding the anti-sense RNA can be from about 20 nucleotides in length up to the length of the relevant mRNA produced by the cell. Preferably, the length of the polynucleotide sequence encoding the anti-sense RNA will be from 50 to 1500 nucleotides in length. The preferred source of anti-sense RNA transcribed from DNA constructs of the present invention is polynucleotide sequences showing substantial identity or similarity to the polynucleotide sequence or fragments disclosed herein.

Using the sequences herein disclosed, it is possible to identify evolutionarily related sequences (referred to as orthologues) in other animals, such as mammals. This may be expected to assist in providing an animal model for schizophrenia, other psychotic disorders and mood disorders associated with the improper functioning of the polynucleotide sequences and/or proteins of the present invention. Once identified, the orthologous sequences can be manipulated in several ways common to the skilled person in order to alter the functionality of the polynucleotide sequences and proteins. For example, "knock-out" animals may be created, that is, the expression of the genes comprising the polynucleotide sequences orthologous to those of the present invention may be reduced or substantially eliminated, in order to determine the effects of reducing or substantially eliminating the expression of the genes. Alternatively, animals may be created where the expression of the polynucleotide sequences and proteins orthologous to those of the present invention are upregulated, that is, the expression of the genes comprising the polynucleotide sequences orthologous to those of the present invention may be increased, in order to determine the effects of increasing the expression of these genes. In addition to these manipulations, substitutions, deletions and additions may be made to the polynucleotide sequences encoding the proteins orthologous to those of the present invention in order to effect changes in the activity of the proteins to help elucidate the function of domains, amino acids, etc in the proteins. Furthermore, the sequences of the present invention may also be used to transform animals in the manner described above. The manipulations described above may also be used to create an animal model of schizophrenia, other psychotic disorders and mood disorders associated with the improper functioning of the polynucleotide sequences and proteins of the present invention which, in turn, may be used to screen for and/or evaluate potential or candidate agents which may be effective for combating schizophrenia, other psychotic disorders and mood disorders. Therefore, in a still further aspect, the present invention provides a transgenic animal which comprises novel sequences according to the present invention. The transgenic animal may, for example, be a mammal such as a laboratory animal.

In a further aspect, the present invention provides a kit for identifying a candidate agent which modulates the interaction between a DISC1 partner polypeptide and DISC1 polypeptide, said kit comprising:

(a) a DISC1 polypeptide, splice variants, fragments, or orthologues thereof; and (b) a DISC1 partner polypeptide selected from the group consisting of ATF4,

KIAA1167, KIAA0380, AKAP450 and NUDE1 , or splice variants/mutant, fragments, or orthologues thereof.

The kit may include detection means for detecting any modulation in the binding of said DISC1 partner to said DISC1 polypeptide in the presence of the candidate agent. However, the detection means may not be included when using, for example, a gel retardation assay to detect any modulation.

The present invention further provides a method of screening for a candidate agent which modulates the interaction between a DISC1 partner polypeptide and DISC1 polypeptide, said method comprising: a) introducing into a suitable yeast host cell an expression vector comprising a polynucleotide fragment encoding DISC1 , or a fragment, derivative or homologue thereof, fused to the

DNA binding domain of the yeast transcription factor GAL4; b) introducing into the same yeast host cell an expression vector comprising a polynucleotide fragment encoding any one of the DISC1 partners disclosed herein, or a fragment, derivative or homologue thereof, fused to the activation domain of the yeast transcription factor GAL4; c) bringing the host cell from step b) into contact with potential modulatory agents, by growth on medium containing said compounds and peptides; and d) determining whether the agent has influenced the interaction by means of measuring the degree of reporter gene activation.

An example of a suitable reporter gene may be, but is not restricted to, the reporter genes HIS3, URA3 and lacZ. The present invention also provides a method of screening for a candidate agent which modulates the interaction between a DISC1 partner polypeptide and DISC1 polypeptide, said method comprising: a) coupling in vitro transcription/ translation of a polynucleotide fragment encoding DISC1 , or a fragment, derivative or homologue thereof, fused to glutathione-S-transferase (GST); b) incubating the GST-DISC1 fusion from step a) with glutathione-agarase beads to allow binding to occur; c) coupling in vitro transcription/ translation of a polynucleotide fragment encoding any one of the DISC1 partners disclosed herein, or a fragment, derivative or homologue thereof, in the presence of [³⁵S] methionine; d) mixing the GST-DISC1 fusion bound to agarose beads and the radiolabelled DISC1 partner in the presence of potential modulatory agents; e) isolating agarose beads and associated polypeptides from the reaction; determining whether the agent has influenced the interaction by analysis of the degree of association of radiolabelled DISC1 partner with the agarose beads. In a still further aspect, the present invention provides an agent identified by any one or more of the assays disclosed herein. The agents may be useful in reducing or substantially eliminating the symptoms of schizophrenia, other psychotic disorders and mood disorders. The identified agents may be prepared with a pharmaceutically acceptable carrier.

For all pharmaceutical formulations disclosed herein, pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0 0.1M and preferably 0.05M phosphate buffer or 0.8% saline. Additionally, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like.

As discussed herein, mutations in the polynucleotide sequences of any one of the DISC1 partners may affect the binding of the partners to DISC1 , and such mutations may be involved in schizophrenia, other psychotic disorders and mood disorders or predispose an individual to developing schizophrenia, other psychotic disorders and mood disorders. Therefore, detection of such mutations may assist in diagnosing or determining an individual's susceptibility to schizophrenia, other psychotic disorders and mood disorders. Thus, the present invention provides a method of diagnosing schizophrenia, other psychotic disorders and mood disorders in an individual or the potential for an individual to develop such a disorder, said method comprising determining any changes in the polynucleotide or polypeptide sequences encoding any one of the DISC1 partners.

The term "schizophrenia, other psychotic disorders and mood disorders" as used herein relates to schizophrenia, schizotypal and delusional disorders, as well as other affective psychoses as listed in "The ICD-10 Classification of Mental and Behavioural Disorders" World Health Organization, Geneva 1992. Categories F20 to F29 inclusive includes Schizophrenia, schizotypal and delusional disorders. Categories F30 to F39 inclusive are Mood (affective) disorders that include manic episode bipolar affective disorder, depressive episode, recurrent depressive disorder, persistent mood disorder, other mood disorders and unspecified mood disorder. Additionally and alternatively, schizophrenia, other psychotic disorders and mood disorders as used herein relate to schizophrenia, other psychotic disorders and mood disorders as listed in the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV). American Psychiatric Association, Washington DC. 1994. Schizophrenia, other psychotic disorders and mood disorders includes all sections of the diagnostic codes 293, 295, 297, 298. Mood disorders includes depressive disorders and bipolar disorders described in all sections of the diagnostic code 296, 300.40, 311 and 301.13, including major depressive disorder, dysthymic disorder, bipolar I disorder, bipolar II disorder and cyclothymic disorder.

Analysis of expressed sequence tags deposited in the Unigene database

(http://www.ncbi.nlm.nih/gov) suggested that KIAA1167, AKAP450 and NUDE1 expression may be detectable in blood while ATF4 and KIAA0380 expression may be detectable in blood and skin. Other tissues include brain tissue which may be obtained post-mortem for epidemiological purposes.

Detection/diagnosis methods may include, but are not limited to, quantitative reverse- transcription PCR (compares relative RNA levels between affecteds and unaffecteds, and detects alterations in the balance of alternative splicing), reverse-transcription PCR (may identify transcripts containing deletions and insertions). The detection/diagnosis methods also include mutation detection methods such as PCR and SSCP, PCR and sequencing, and PCR snapshot. The presence and/or levels of the DISC1 partners polypeptides may be determined using immunological methods such as radioimmunoassays and ELISA assays (which compare polypeptide expression levels between affecteds and unaffecteds) and western blots (which may be used to identify abnormal polypeptides which may be oversized or truncated, or over/under-expression of polypeptides, or changes in the balance of alternatively spliced isoforms). Antibodies may also be designed and used to detect the presence of abnormal DISC1 partner polypeptides compared with normal polypeptides. Therefore, the present invention also encompasses antibodies raised to epitopes encoded by alternatively spliced exons or mutated forms of any one of the DISC1 partners disclosed herein and antibodies to native/normal forms of these proteins, where they have not previously been disclosed. Production and purification of antibodies specific to an antigen is a matter of ordinary skill, and the methods to be used are clear to those skilled in the art. The term antibodies can include, but is not limited to, polyclonal antibodies, monoclonal antibodies (mAbs), humanised or chimeric antibodies, single chain antibodies, Fab fragments, (Fab')₂ fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope binding fragments of any of the above. Such antibodies may also be used as agents to affect the binding of any one of the DISC1 partners to the DISC1 polypeptide and, as such, may be used in therapy.

If mutations in the polynucleotide sequences of any one of the DISC1 partners are identified in an individual, it may be suitable to treat such an individual to alleviate or substantially eliminate the symptoms of the schizophrenia, other psychotic disorders and mood disorders. This may be performed by administering a copy of the relevant gene which does not have any mutations, i.e. a "healthy gene". This may be typically done using standard gene therapy techniques commonly known in the field. Therefore, the present invention further provides a method of treating an individual with schizophrenia, other psychotic disorders and mood disorders or predisposed to developing schizophrenia, other psychotic disorders and mood disorders, said method comprising administering to said individual a polynucleotide fragment comprising a polynucleotide sequence of any one or more of the DISC1 partner polynucleotide sequences disclosed herein, or a fragment, derivative, or orthologue thereof. As used herein, the term "gene therapy" is understood to mean the introduction into a cell or cells of an exogenous polynucleotide fragment for the purpose of treating a disorder and/or abnormality. The disorder and/or abnormality may be genetic or otherwise.

Alternatively, it may be suitable to administer to the individual a polypeptide, or a fragment or domain, derived from any one or more of the DISC1 partner sequences to alleviate or substantially eliminate the symptoms of the schizophrenia, other psychotic disorders and mood disorders. Thus, the present invention also provides a method of treating an individual with schizophrenia, other psychotic disorders and mood disorders or predisposed to developing schizophrenia, other psychotic disorders and mood disorders, said method comprising administering to said individual a polypeptide comprising an amino acid sequence of any one or more of the DISC1 partner amino acid sequences disclosed herein, or a functionally active fragment, derivative, or orthologue thereof.

Once a particular polymorphism or mutation has been identified using the information provided herein, it may be possible to optimise a particular course of treatment. For example it is known that some patients respond to certain medication while others do not. Additionally, some patients respond adversely to particular forms of treatment while others do not. This may in fact be due to the nature of the mutations in the gene or surrounding sequence, and it may therefore be possible to optimise a treatment strategy using current and/or prospective therapies, based on a patient's genotype, and avoid adverse reactions.

With references to polypeptides, fragments are defined herein as any portion of the polypeptide described herein that substantially retains the activity of the full-length polypeptide or of any functional domains of the polypeptide. Derivatives are defined as any modified forms of the polypeptide which also substantially retains the activity of the full-length polypeptide. Such derivatives may take the form of amino acid substitutions which may be in the form of like for like e.g. a polar amino acid residue for another polar residue or like for non-like eg. substitution of a polar amino acid residue for a non-polar residue as discussed in more detail below.

Replacement amino acid residues may be selected from the residues of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. The replacement amino acid residue may additionally be selected from unnatural amino acids. Within the above definitions of the peptide carrier moieties of the present invention, the specific amino acid residues of the peptide may be modified in such a manner that retains their ability to induce apoptosis, such modified peptides are referred to as "variants". Thus, homologous substitution may occur i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar, etc. Non-homologous substitution may also occur i.e. from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (O), diaminobutyric acid (B), norleucine (N), pyriylalanine, thienylalanine, naphthylalanine and phenylglycine and the like. Within each peptide carrier moiety, more than one amine acid residue may be modified at a time, but preferably, when the replacing amino acid residue is alanine, less than 3.

As used herein, amino acids are classified according to the following classes; basic; H,K,R " acidic; D,E polar, A,F,G,I,L,M,PN,W non-polar; C,N,Q,S,T,Y,

(using the internationally accepted amino acid single letter codes) and homologous and non-homologous substitution is defined using these classes. Thus, homologous substitution is used to refer to a substitution from within the same class, whereas non-homologous substitution refers to a substitution from a different class or by an unnatural or synthetic amino acid.

In general, the term "polypeptide" refers to a molecular chain of amino acids with a biological activity, and is used herein interchangeably with the term "protein". It does not refer to a specific length of the products, and if required it can be modified in vivo and/or in vitro, for example by glycosylation, myristoylation, amidation, carboxylation or phosphorylation; thus inter alia peptides, oligopeptides and proteins are included. The polypeptides disclosed herein may be obtained, for example, by synthetic or recombinant techniques known in the art.

It will be understood that for the DISC1 partner polynucleotide and polypeptide sequences referred to herein, natural variations can exist between individuals. These variations may be demonstrated by amino acid differences in the overall sequence or by deletions, substitutions, insertions or inversions of amino acids in said sequence. All such variations are included in the scope of the present invention.

Over-expression or under-expression of any one of the DISC1 partners may affect the downstream activity of DISC1, and any such changes in expression may be involved in schizophrenia, other psychotic disorders and mood disorders. Therefore, upregulating expression of a DISC1 partner when the partner is being under-expressed, or downregulating expression of a DISC1 partner when the partner is being over-expressed may reduce or substantially eliminate the symptoms of the schizophrenia, other psychotic disorders and mood disorders. Thus, in a further aspect, the present invention provides a method of screening for and evaluating an agent which affects the expression of the proteins of the present invention, said method comprising administering the agent to a cell line or animal wherein the levels of expression of any one of the DISC1 partners has been altered, and determining the effect of said agent on the expression of the proteins of the present invention. The polynucleotide sequence of the DISC1 partner may be the native/normal sequence i.e. unmutated, or may be mutated.

In a yet further aspect, the present invention provides use of the polynucleotide sequences disclosed herein or sequences complementary to said polynucleotide sequences for use in determining a loss of expression of any one of the DISC1 partners. Such a loss may be determined using techniques such as northern blot analysis, RT-PCR, micro arrays, DNA arrays or DNA chips and other techniques known in the art.

In a still further aspect, the present invention provides use of a polynucleotide fragment comprising a polynucleotide sequence of any one of the DISC1 partners disclosed herein, or a fragment, derivative, or orthologue thereof, in the manufacture of a medicament for the treatment of schizophrenia, other psychotic disorders and mood disorders. The schizophrenia, other psychotic disorders and mood disorders may, for example, be schizophrenia.

In a yet further aspect, the present invention provides use of a polypeptide comprising an amino acid sequence of any one of the DISC1 partners disclosed herein, or a functionally active fragment, derivative, or orthologue thereof, in the manufacture of a medicament for the treatment of schizophrenia, other psychotic disorders and mood disorders. The schizophrenia, other psychotic disorders and mood disorders may, for example, be schizophrenia.

The amino acid sequences (polypeptide or protein) disclosed herein may be purified from in vivo sources or may be prepared by recombinant or synthetic techniques common to the skilled person.

In a yet further aspect, the present invention provides a pharmaceutical formulation comprising a polynucleotide fragment comprising a polynucleotide sequence of any one of the DISC1 partners disclosed herein, or a fragment, derivative, or orthologue thereof, and a pharmacologically acceptable carrier. In a still further aspect, the present invention provides a pharmaceutical formulation comprising a polypeptide comprising an amino acid sequence of any one of the DISC1 partners disclosed herein, or a functionally active fragment, derivative, or orthologue thereof, and a pharmacologically acceptable carrier. ln a yet further aspect, the present invention provides a kit for detecting one or more mutations in a gene sequence encoding a DISC1 partner selected from the group consisting of ATF4, KIAA1167, KIAA0380, AKAP450 and NUDE1 , said kit comprising:

(a) oligonucleotides capable of binding to the gene of a DISC1 partner; and (b) necessary reagents.

Detection of the mutations may be made using DNA sequencing, hybridisation, or the like. The oligonucleotides correspond to normal DNA sequences which may flank any mutated sequences, or may correspond to the mutated DNA sequences to be detected.

These and other aspects of the present invention will become apparent from the following description when taken in combination with the accompanying drawings, in which:

Figure 1A illustrates the polynucleotide sequence of ATF4;

Figure 1B illustrates the amino acid sequence of ATF4;

Figure 1C illustrates a sequence alignment between ATF4 sequences from various species, with the DISC1 polypeptide interacting sequence indicated (full species names for this figure and the following figures are Pan troglodytes, Gallus gallus, Rattus norvegicus, Mus musculus, Oryctolagus cuniculus, Papio cynocephalus, Tetraodon nigroviridis, Felis catus, Bos taurus, Canis familiaris, Sus scrofa and Takifugu Rubripes);

Figure 2A illustrates the polynucleotide sequence of KIAA1167; The open reading frame specified by the polynucleotide sequence with GenEMBL accession number AB032993 was incomplete at the 5' end. The open reading frame was completed utilising the polynucleotide sequence with GenEMBL accession number BF724120.

Figure 2B illustrates the amino acid sequence of KIAA1167;

Figure 2C illustrates a sequence alignment between KIAA1167 sequences from various species, with the DISC1 polypeptide interacting sequence indicated; Figure 3A illustrates the polynucleotide sequence of a first splice variant of KIAA0380 lacking exon 34, with the site of inclusion of exon 34 located between underlined bases 1327 and 1328; Figure 3B illustrates the amino acid sequence of the first splice variant KIAA0380 arising from transcripts lacking exon 34, with the position of amino acids encoded by exon 34 located between underlined amino acids 195 and 196;

Figure 3C illustrates the polynucleotide sequence of a second, alternate splice variant of KIAA0380, referred to as KIAA0380alt utilising exon 34, with the exon 34 sequence indicated in bold;

Figure 3D illustrates the amino acid sequence of KIAA0380 arising from transcripts utilising exon 34, with the amino acids encoded by exon 34 indicated in bold;

Figure 3E illustrates a sequence alignment between KIAA0380 sequences from various species, with the DISC1 polypeptide interacting sequence and the exon 34 sequence indicated;

Figure 3F illustrates a schematic of KIAA0380 genomic structure: black vertical bars indicate exons previously identified as belonging to KIAA0380 on the human genome browser, the alternatively spliced exon 34 is indicated in grey. (A) all exons, alternative exon in grey, and (B) as annotated in human genome browser; Figure 4A illustrates the polynucleotide sequence of a first splice variant of AKAP450, referred to as AKAP450alt, utilising exon 1 b, with the exon 1 b sequence indicated in bold and the translation initiation codon is indicated by the white text in a black box;

Figure 4B illustrates the amino acid sequence of a first splice variant of AKAP450 (AKAP450alt), predicted to result from utilisation of exon 1 b, with the alternative sequence indicated in bold type;

Figure 4C illustrates the polynucleotide sequence of a second, alternate splice variant of AKAP450, referred to as AKAP450wexon28a which utilises exon 28a, with the site of alternative splicing in exon 28 is found between the underlined bases 5826 and 5827;

Figure 4D illustrates the amino acid sequence of a second, alternate splice variant of AKAP450 (AKAP450 wexon28a), predicted to result from utilisation of exon 28a, with the site of alternative splicing in exon 28 found between the underlined amino acids 2162 and 2163;

Figure 4E illustrates the polynucleotide sequence of a third, alternate splice variant of AKAP450 which utilises exon 28, with the site of inclusion of exon 1 b found between the underlined bases 5826 and 5827; Figure 4F illustrates the amino acid sequence of a third, alternate splice variant of AKAP450, predicted to result from utilisation of exon 28, with the site of inclusion of exon 1 b found between the underlined amino acids 1868 and 1869;

Figure 4G illustrates a sequence alignment between AKAP450 sequences from various species, with the DISC1 polypeptide interacting sequence and the amino acids affected by exon 28 alternative splicing indicated;

Figure 4H illustrates a schematic of AKAP450 genomic structure: black vertical bars indicate exons previously identified as belonging to AKAP450 on the human genome browser, the alternatively spliced exons are indicated in grey. (A) all exons, alternative exons in grey, and (B) as annotated in human genome browser;

Figure 5A illustrates the polynucleotide sequence of a first splice variant of NUDE1, referred to as mNUDEa utilising exon 9a, with the exon 9a sequence indicated in bold, the internal splice acceptor site is found between the underlined bases (utilisation of this splice site produces exon 9b) and the stop codon is indicated by the white text in a black box; Figure 5B illustrates the amino acid sequence of the first splice variant of NUDE1

(mNUDEIa), with the amino acids derived from the alternately spliced polynucleotide sequence indicated in bold;

Figure 5C illustrates the polynucleotide sequence of a second, alternate splice variant of NUDE1 utilising exon 9b, referred to as NUDEI b, with the exon 9b sequence indicated in bold and the stop codon is indicated by the white text in a black box;

Figure 5D illustrates the amino acid sequence of the second, alternate splice variant of NUDE1 (mNUDEI b), with the amino acids derived from the alternately spliced polynucleotide sequence indicated in bold;

Figure 5E illustrates the polynucleotide sequence of a third, alternate splice variant of NUDE1 utilising exon 9c, referred to as NUDE1 c, with the exon 9c sequence indicated in bold and the stop codon is indicated by the white text in a black box;

Figure 5F illustrates the amino acid sequence of the third, alternate splice variant of NUDE1 (NUDEI c), with the amino acids derived from the alternately spliced polynucleotide sequence indicated in bold; Figure 5G illustrates a sequence alignment between NUDEIb sequences from various species, with the DISC1 polypeptide interacting sequence indicated;

Figure 5H illustrates a schematic of the NUDE1 genomic structure: vertical bars indicate exons, black bars indicate exons previously identified as belonging to NUDE1 on the human genome browser and the alternative exon 9s are indicated in grey. The final exon (9c) overlaps with exons of the MYH11 gene, while exon 9a/b contains an internal splice acceptor site such that two forms of this exon may be included in transcripts. (A) all exons illustrated, (B) NUDE1 as annotated in the human genome browser, (C) an alternative 3' UTR of NUDE1, and (D) an alternative 3' UTR of NUDE1 orthologous to other species; Figure 5i shows a reciprocal translocation in chromosomes 1 and 16 in a patient with schizophrenia;

Figure 5j shows in more detail the effect of a breakpoint on chromosome 16 as shown in Figure 5i; and

Figure 5k shows the relative expression patterns of the genes in the vicinity of the chromosome 16 breakpoint.

EXAMPLES Methods

Vector Construction A full length human DISC1 ORF was amplified by PCR using Advantage-GC cDNA polymerase mix (Clontech) and human heart cDNA as template, inserted into pDBLeu (GIBCO BRL) and fully sequenced to confirm sequence integrity. This construct is referred to as pDBLeuDISCI .

Yeast Two-Hybrid Library Screen

Library-scale frozen competent MaV203 yeast cells (GIBCO BRL) were co-transformed with pDBLeuDISCI plus PROQUEST human adult or foetal (third trimester) brain cDNA libraries (constructed in pPC86, GIBCO BRL) according to the manufacturers instructions. Transformed cells were plated on selective plates containing 50 mM 3-AminoTriazole according to the manufacturers instructions. Colonies surviving selection were assayed for the presence of proteins interacting with DISC1 according to the manufacturers instructions.

Identification of Interacting Proteins Plasmids expressing interacting proteins were isolated from yeast and used to transform electro-competent Escherichia coli strain DH10B. Plasmids were isolated from bacterial transformants and sequenced using primers flanking the pPC86 multiple cloning site (forward = tataacgcgtttggaatcact, reverse = gtaaatttctggcaaggtagac), Sequences were used to query the EMBL, TrEMBL and Swissprot polynucleotide and protein sequence databases using appropriate versions of BLAST at the Medical Research Council United Kingdom Human Genome Mapping Project Resource Centre.

Confirmation of Interactions in the Yeast Two-Hybrid System

To confirm that the interacting proteins had been correctly identified, competent MaV203 yeast cells were sequentially transformed with pDBLeuDISCI followed by each plasmid expressing a putative interactor. Colonies were assayed for DISC1 -protein interactions according to the manufacturers instructions. If the plasmid failed to express an interacting protein, several further E. coli transformants derived from the original yeast colony were sequenced, and assayed for interactions with DISC1 as appropriate, until the interacting protein was identified.

Results

Screening a total of 700,000 transformants from the adult brain library, and 350,000 transformants from the foetal brain library revealed six proteins that bind very strongly to DISC1 in yeast. Each of these proteins is critical within the central nervous system, suggesting that DISC1 , in turn, is also essential to the development and functioning of the brain. Further weight is thus added to the body of evidence pointing towards involvement of DISC1 in the aetiology of schizophrenia. ATF4

Seven independent clones encoded ATF4, also known as CREB2. Clones pPC86.5, 7 and 17, and FB11 , 12, 8 and 5 encode amino acids 38-stop, 78-stop, 185-stop, 194-stop, 235- stop, 250-stop, and 285-stop, respectively. ATF4 is a cAMP-dependent transcription factor that is involved in many non-neuronal functions. However, in the central nervous system it may be important in the process of memory formation since in the sea snail Aplysia the homologue apCREB2 is involved in the process of synaptic plasticity (Bartsch et al, Cell 103: 595-608, 2000). Additionally, in rat and human neurons, ATF4 interacts directly with GABA_B receptors in the cytoplasm, and translocates between the cytoplasm and nucleus in response to receptor stimulation (White et al, PNAS 97, 13967-13972, 2000, Nehring et al, J Biol Chem 275, 35185- 35191 , 2000, Meyer et al, Mol Cell Neurosci 17, 637-645, 2001), suggesting involvement in a novel signalling mechanism for activity-dependent gene expression.

ATF4 is located less than 3.5 Mb from D22S278, a marker consistently providing evidence for linkage between 22q13 and schizophrenia or bipolar affective disorder. The maximum LOD score obtained to date is 3.8 for bipolar affective disorder (Kelsoe et al, PNAS 98: 585-590). Hence ATF4 may itself contribute to the aetiology of psychiatric illness as well as mediating the pathological effects of DISC1 mutations.

KIAA1167 Two separate clones encoding protein KIAA1167 (now also referred to as GRASP1) were identified. Clone pPC86.22 encodes amino acids 312-stop of KIAA1167, while clone pPC86.24 encodes amino acids 442-stop. KIAA1167 shows at least 94% identity with rat GRASP-1 and is consequently likely to be the human orthologue of this protein. In rat, GRASP- 1 is expressed in neurons, interacting directly with the adapter protein GRIP (Ye et al, Neuron 26: 603-617, 2000). GRIP interacts directly with AMPA receptors and GRASP-1 is complexed with these proteins in rat brain (Ye et al, Neuron 26: 603-617, 2000). GRASP-1 may control AMPA receptor targeting to the synapse and regulation of this process by NMDA receptors (Ye et al, Neuron 26: 603-617, 2000). GRASP-1 is a member of the rasGEF family of proteins that activate the intracellular messenger ras to regulate a variety of processes including cell growth, differentiation, transformation and NMDA-dependent synaptic plasticity (Ye et al, Neuron 26: 603-617, 2000). These rasGEFs are important members of signal transduction complexes and GRASP-1 may be recruited to these complexes by GRIP. In addition to the AMPA receptor complex, this recruitment process may also include ephri signalling complexes (Ye et al, Neuron 26: 603- 617, 2000), since GRIP interacts with ephrin B ligands (Lin et al, J Biol Chem 274: 3726-3733, 1999, Bruckner et al, Neuron 22: 511-524, 1999) and eph receptors (Torres et al, Neuron 21 : 1453-1463, 1998). Ephrins and their receptors act as positional markers required for processes such as axon guidance and directing migrating neurons (for review see Flanagan & Vanderhaeghen, Ann Rev Neurosci 21 : 309-345, 1998).

The KIAA1167 gene is located at Xp11 , where there is weak evidence for linkage to schizophrenia and related psychiatric disorders (Dann et al, Psychiatry Res 70: 131-143, 1997). The human orthologue of GRASP-1 may therefore contribute to the aetiology of psychiatric illness as well as mediating the pathological effects of DISC1 mutations.

KIAA0380

A single clone, pPC86.9, encoded amino acids 1354-stop of protein KIAA0380, also known as PDZ-RhoGEF or ARHGEF11. KIAA0380 is approximately 85% identical to rat GTRAP48 at the amino acid level, indicating that these proteins are probable orthologues. GTRAP48 is brain-specific and interacts directly with the neuronal glutamate transporter EAAT4 (Jackson et al, Nature 410: 89-93). Interaction with GTRAP48 stabilises EAAT4 at the cell membrane and enhances glutamate transport activity (Jackson et al, Nature 410: 89-93).

KIAA0380 and GTRAP48 possess guanine polynucleotide exchange factor activity and activate Rho to induce actin cytoskeleton reorganisation (Jackson et al, Nature 410: 89-93, Fukuhara et al, J Biol Chem 274: 5868-5879, 1999, Rumenapp et al, FEBS Lett 459: 313-318, 1999). KIAA0380 apparently regulates neurite retraction through this mechanism of Rho- dependent signalling (Togashi et al, J Biol Chem 275: 29570-29578, 2000). Furthermore KIAA0380 and GTRAP48 bind to activated G subunits from the heterotrimeric G protein complex (Jackson et al, Nature 410: 89-93, Fukuhara et al, J Biol Chem 274: 5868-5879, 1999), suggesting that they may transduce cell surface signals from G-protein coupled receptors to the actin cytoskeleton via Rho.

The gene encoding KIAA0380 is located at 1q23 within a region of the human genome implicated as a major schizophrenia susceptibility locus (LOD=6.5, Brzustowicz et al, Science

288: 678-682, 2000). Consequently, this gene may confer susceptibility to schizophrenia in a significant proportion of individuals, in addition to mediating the pathological effects of DISC1 mutations.

AKAP450

Clone pPC86.3 encodes amino acids 2141-2629 of the multiply spliced human protein AKAP450, also referred to as AKAP9. CG-NAP and AKAP350 refer to smaller alternatively spliced versions (Takahashi et al, J Biol Chem 274: 17267-17274, 1999, Schmidt et al, J Biol Chem 274: 3055-3066, 1999). Yotiao, claimed to be another shorter isoform arising from alternative splicing, is a component of the post-synaptic density, interacting with the NR1 subunit of NMDA receptors, the Rll subunit of protein kinase A (PKA) and with protein phosphatase 1 (PP1 , Lin et al, J Neurosci 18: 2017-2027, 1998, Feliciello et al, FEBS Lett 464: 174-178, 1999, Westphal et al, Science 285: 93-96, 1999). Yotiao apparently links PKA and PP1 to NMDA receptors to regulate channel activity (Westphal et al, Science 285: 93-96, 1999). CG-NAP interacts with PKA and PP1 , as well as protein kinase N, protein phosphatase

2A and protein kinase C (Takahashi et al, J Biol Chem 274: 17267-17274, 1999, Takahashi et al, J Biol Chem 275: 34592-34596, 2000). In non-neuronal cells CG-NAP and AKAP450 are present at the centrosome and are presumed to act as a 'scaffold' for assembly of these kinases and phosphatases (Takahashi et al, J Biol Chem 274: 17267-17274, 1999, Witczak, et al, EMBO 318: 1858-1868, 1999).

The stretch of amino acids encoded by clone pPC86.3 is not contained within the sequence reported for yotiao and therefore it would seem that an involvement of DISC1 in regulation of NMDA receptor activity via yotiao is unlikely. However, the in vivo studies of yotiao at the post-synaptic density carried out to date would not distinguish yotiao from other isoforms or the full-length AKAP450. Furthermore, the major transcript in brain apparently corresponds to the full-length protein rather than yotiao, while the major protein species extracted from brain is substantially larger than yotiao (Lin et al, J Neurosci 18: 2017-2027, 1998, Schmidt et al, J Biol Chem 274: 3055-3066, 1999, Witczak, et al, EMBO 318: 1858-1868, 1999). Consequently it is a distinct possibility that the full-length protein or AKAP350 isoforms may interact with NMDA receptors, and DISC1 may therefore also be a component of the post-synaptic density.

The gene encoding AKAP450 and its alternatively spliced isoforms is located at 7q21 close to a region of the human genome implicated as a schizophrenia susceptibility locus

(LOD=3.18, Ekeiund et al, Hum Mol Genet 9:1049-1057, 2000). Consequently, this gene may contribute to the aetiology of schizophrenia, in addition to mediating the pathological effects of

DISC1 mutations.

NUDE1 (formerly referred to as mNUDE)

A single clone, pPC86.20 encodes the entire ORF of an apparently abundant variant of NUDE1 , where the final exon is antisense to three exons of MYH11 (Human Genome Project Working Draft Human Genome Browser http://genome.ucsc.edu/). NUDE1 interacts with LIS1 (Sasaki et al, Neuron 28: 681-696, 2000, Niethammer et al, Neuron 28: 697-711 , 2000, Feng et al, Neuron 28: 665-679, 2000). Mutations in LIS1 result in the human neuronal migration disorder isolated lissencephaly sequence (ILS, Reiner et al, Nature 364: 717-721 , 1993) and ILS mutations block the interaction between LIS1 and NUDE1 (Sasaki et al, Neuron 28: 681- 696, 2000, Feng et al, Neuron 28: 665-679, 2000). Furthermore, Xenopus, overexpression of the NUDE1 LIS1-binding domain perturbs central nervous system lamination and architecture (Feng et al, Neuron 28: 665-679, 2000).

In cultured neurons NUDE1 is present at the centrosome (Sasaki et al, Neuron 28: 681- 696, 2000, Niethammer et al, Neuron 28: 697-711 , 2000, Feng et al, Neuron 28: 665-679, 2000), interacts with γ-tubulin and may regulate microtubule organisation (Feng et at, Neuron 28: 665-679, 2000).

LIS1 , dynein and NUDE1 are orthologues of fungal proteins, nudF, nudA and nudE respectively, all involved in nuclear migration (Xiang et al, Mol Biol Cell 6: 297-310, 1995, Xiang et al PNAS 91 : 2100-2104, 1994, Efimov & Morris J Cell Biol 150: 681-688, 2000). This pathway is highly conserved and related to the process of neuronal migration, where a migrating cell first extends a leading process along the radial glia, followed by repositioning of the nucleus to its final location, 'nucleokinesis', and finally, trailing edge retraction. NUDE1 might play a critical role in the nucleokinesis stage of nuclear migration, in addition to having a vital role in other cellular processes such as axonal transport.

NUDE1 is a paralogue of NUDEL. The proteins encoded by these genes are 64% identical, 79% similar. A robust interaction between exogenously expressed NUDEL and DISC1 in HEK 293 cells has been demonstrated (Ozeki et al, Disrupted-in-Schizophrenia-1 (DISC-1): Mutant truncation prevents binding to NudE-like (NUDEL) and inhibits neurite outgrowth, PNAS 2003, 100: 289-294), with amino acids 201-280 of NUDEL containing the DISC1 interaction site. These amino acids also contain a region of 24 amino acids that are identical with NUDE1 , suggesting that this stretch contains the DISC1 interaction site in both NUDE and NUDEL. Consequently, this demonstration of interaction between DISC1 and NUDEL in mammalian cells provides further support to the interaction between NUDE1 and DISC1.

Evidence exists for linkage between the region of the human genome on 16p13 containing the mNUDE gene and major psychosis (LOD=1.8-2.52, Detera-Wadleigh et al, Am J Med Genet 88: 255-259, 1999). Consequently, this gene may contribute to the aetiology of major psychiatric illness, in addition to mediating the pathological effects of DISC1 mutations.

Further evidence in relation to NUDE1 disruption

A patient with chronic schizophrenia carries a balanced translocation. The reciprocal translocation has two breakpoints; on chromosome 1 and 16 (see Figure 51). The chromosome 1 breakpoint appears to be within the centromeric heterochromatin of the long (q) arn. Hence it is not likely to be either directly disrupting or in close proximity to a gene. The chromosome 16 breakpoint has been mapped to the resolution of bacterial artificial chromosome (BAG) clones. Overlapping clones RP11-629e8 and RP11-844e20 both contain the breakpoint. Examination of the human genome sequence corresponding to these clones indicates that the breakpoint lines in the vicinity of two genes; FU31153 (a novel gene with no known function) and MYH11.

MYH11 encodes smooth muscle myosin, heavy chain 1 , a protein involved in muscle contraction. This gene is also involved in a relatively common pericentric inversion of chromosome 16 which results in the creation of a fusion gene with oncogenic potential - this frequently results in acute myeloid leukemia, most commonly of the M4Eo subtype. The breakpoint described here is most likely to be within the 5' end of the MYH11 gene or immediately upstream of the gene (see Figure 51).

No gene near the chromosome 16 breakpoint is an immediately obvious candidate for psychiatric illness - the expression patterns of the genes are shown in Figure 5k and most are widely expressed. However, in the light of the identification of proteins identified through their interaction with DISC1 , the presence of the NUDE1 gene overlapping the MYH11 gene raised the possibility that the putative MYH11 disruption could perturb the expression of NUDE gene and hence affect the same molecule pathway(s) in which DISC1 acts.

Without wishing to be bound by theory, three possible mechanistic explanations are proposed for this indirect action of the breakpoint on NUDEL

1) The breakpoint may separate the body of the NUDE1 gene from regulatory elements (now translocated to chromosome 1). This would lead to a change in expression pattern or level.

2) The apposition of chromosome 1 heterochromatin and chromosome 16 euchromatin of the derived chromosome 16 may silence or deregulate NUDE1 expression.

This effect is known as "position effect" and has been described in other systems.

3) NUDE1 and MYH11 genes overlap. Therefore there may be competition at the transcriptional (RNApol lll competition) or translational (hybridisation of the two mRNAs preventing ribosome interaction) levels resulting in a steady state of relative gene expression from the two loci. The putative breakpoint-mediated disruption of MYH11 may alter this balance in favour of greater NUDE1 expression. SCREENING METHODS Yeast Two-Hybrid System

Vectors

The vector expressing DISC1 fused to the DNA binding domain of the yeast transcription factor GAL4, referred to as pDBLeuDISCI , has been described herein. Vectors expressing fragments of ATF4, KIAA1167, KIAA0380, AKAP450 and NUDE1 fused to the activation domain of the yeast transcription factor GAL4, referred to as pPC86.5, 7 and 17, and FB11 , 12, 8 and 5 (ATF4), pPC86.22 and 24 (KIAA1167), pPC86.9 (KIAA0380), pPC86.3 (AKAP450) and pPC86.20 respectively, have also been described herein. Where expression of other fragments, derivatives or homologues of these polypeptides is required, ORFs may be amplified by PCR using Advantage-GC cDNA polymerase mix (Clontech) for DISC1 or Proofstart DNA polymerase (Qiagen) for the DISC1 partners, and appropriate cDNA as template. PCR products may be inserted into appropriate vectors designed to express the ORFs fused to the DNA binding or activation domains of the yeast transcription factor GAL4. These vectors may be, but are not restricted to, pDBLeu and pPC86 (Invitrogen). Inserts will be fully sequenced to confirm sequence integrity.

Yeast Transformation

Library-scale frozen competent MaV203 yeast cells (Invitrogen) may be co-transformed with a vector expressing DISC1 , or a fragment, derivative or homologue thereof, fused to the DNA binding domain of the yeast transcription factor GAL4, plus a vector expressing a DISC1 partner, or a fragment, derivative or homologue thereof, fused to the activation domain of the yeast transcription factor GAL4. Transformation may be carried out using standard methods. Transformed cells may be plated on selective plates (synthetic complete medium (13.4g yeast nitrogen base with ammonium sulphate, plus 100ml 40% glucose/ 2 litres) plus essential amino acids (adenine, alanine, arginine, aspartic acid, asparagine, cysteine, glutamate, glutamine, glycine, isoleucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine and valine), plus uracil) containing an appropriate concentration of 3-AminoTriazole, sufficient to suppress reporter gene self-activation. Assay for Reporter Gene Activation in the Presence of Potential Modulatory Agents

Colonies surviving initial selection may be plated on medium containing potential modulatory compounds and peptides and four reporter gene assays can be performed. 1) For assaying activation of the HIS3 reporter gene colonies may be plated on synthetic complete medium plus essential amino acids plus uracil, additionally containing 3- AminoTriazole and the compound or peptide of interest. Colonies surviving this selection maintain the interaction. Where colonies do not survive the selection, the test compound or peptide is likely to have disrupted interaction between DISC1 and the DISC1 partner. 2) For assaying activation of the URA3 reporter gene colonies may be plated on synthetic complete medium containing the compound or peptide of interest, plus essential amino acids, plus histidine. Colonies surviving this selection maintain a strong interaction. Where colonies do not survive selection, the test compound or peptide is likely to have disrupted or weakened interaction between DISC1 and the DISC1 partner. 3) An additional assay for activation of the URA3 reporter gene involves plating the colonies on synthetic complete medium containing the test compound or peptide, plus essential amino acids, plus histidine and uracil, plus 0.2% 5-fluoro-orotic acid (5FOA). Upon activation of

URA3, 5FOA is converted to toxic 5-fluorouracil. Colonies unable to grow under these

^" conditions maintain the interaction, while in colonies surviving selection, the test compound or peptide is likely to have disrupted the interaction between DISC1 and the DISC1 partner.

4) For assaying activation of the LacZ reporter gene colonies may be grown on nitrocellulose filters placed on YPAD medium (10g bacto-yeast extract, 20g bacto-peptone, 20g dextrose, 100mg adenine sulphate/1 litre) containing the compound or peptide of interest. When growth is sufficient the nitrocellulose filters are dipped in liquid nitrogen for 10-20 seconds to lyse the cells, placed on filter paper soaked in a solution consisting of 100 microlitres of 100 mg/ml X-Gal (5-bromo-5-chloro-3-indolyl-D-galactoside) in N,N-dimethylformamide, plus 60 microlitres of 2-mercaptoethanol, plus 10ml of buffer (16.1g Na₂HP0 .7H₂0, 5.5g NaH₂P0₄, 0.75g KCl, 0.246g MgS0₄.7H₂0/litre distilled water, pH7.0) and incubated at 37°C. Activation of the LacZ reporter gene will turn the colonies blue. A qualitative measure of the strength of interaction between DISC1 and the DISC1 partner may be obtained by monitoring speed and intensity of blue colour development. Alteration of these factors indicates that the test compound or peptide is likely to have affected the interaction between DISC1 and the DISC1 partner.

GST Pull-Down Vectors

DISC1 and DISC1 partners ORFs may be amplified by PCR using Advantage-GC cDNA polymerase mix (Clontech) for DISC1 , or Proofstart DNA polymerase (Qiagen) for the DISC1 partners, and appropriate cDNA as template. PCR products may be inserted into appropriate vectors designed to express the ORFs fused to glutathione-S-transferase (GST) or as native polypeptides. Inserts may be fully sequenced to confirm sequence integrity.

Preparation of GST-Fusion Proteins Using the TNT Quick Coupled Transcription/Translation System (Promega) GST-fusion proteins may be generated in vitro. The products of the coupled in vitro transcription translation may be mixed with glutathione-agarose beads and incubated for approximately 2 minutes at room temperature. The beads with bound protein may then be pelleted and washed twice in phosphate buffered saline.

Generation of Test Proteins

³⁵S-labelled test protein may be generated from appropriate expression vectors using the TNT Quick Coupled Transcription/Translation System (Promega) in the presence of 35^s- labelled methionine,

GST Pull-Down

Known quantities of the GST fusion protein bound to agarose beads may be mixed with known quantities of the ³⁵S-methionine-labelled test protein in the presence of potential modulatory compounds or peptides. The mix may be incubated 1-2 hours at 4°C with gentle mixing. The beads may be pelleted by microcentrifugation, then washed three times in buffer (50mM potassium phosphate, 150mM KCl, 1 mM MgCI₂, 10% glycerol and 1% Triton X-100). The beads may then be electrophoresed through an agarose gel, which may subsequently be fixed and dried. The gel may then be autoradiographed and quantitated by densitometry to determine the quantity of ³⁵S-methionine-labelled test protein pulled down by the bait protein fused to GST. Because this method is quantitative, it can be used as a measure of the strength of protein binding and is likely to identify any increases or decreases in the strength of interactions due to the presence of modulatory compounds or peptides.

It is to be understood that the above is merely exemplary and is not to be construed as limiting in any way.

Claims

1. In a first aspect, the present invention provides a method of screening for a candidate agent which modulates an interaction between a DISC1 partner polypeptide and DISC1 polypeptide, said method comprising:

(a) forming a mixture comprising the candidate agent, a DISC1 partner polypeptide, selected from the group consisting of ATF4, KIAA1167, KIAA0380, AKAP450 and mNUDE/NUDE1 , or splice variants, mutant, fragments, or orthologues thereof, and DISCI polypeptide; (b) subjecting the mixture to conditions suitable for allowing a native DISC1 partner polypeptide and a native DISC1 polypeptide to bind to one another in the absence of any agent; and

(c) detecting any modulation in the binding of said DISC1 partner to said DISC1 polypeptide.

2. The method according to claim 1 wherein the polypeptide sequence of ATF4 is substantially as shown in Figure 1 B or fragments thereof which is capable of interacting with DISC1.

3. The method according to claim 1 wherein the polypeptide sequence of KIAA1167 is substantially as shown in Figure 2B, or fragments thereof which is capable of interacting with DISC1.

4. The method according to claim 1 wherein the polypeptide sequence of KIAA0380 is substantially as shown in Figures 3B or 3D, or fragments thereof which is capable of interacting with DISC1.

5. The method according to claim 1 wherein the polypeptide sequence of AKAP450 is substantially as shown in Figures 4B, 4D or 4F, or fragments thereof which is capable of interacting with DISC1.

6. The method according to claim 1 wherein the polypeptide sequence of NUDE1 is substantially as shown in Figures 5B, 5D or 5F, or fragments thereof which is capable of interacting with DISC1.

7. The method according to any preceding claim wnerem saiα tragment corresponds to a polypeptide domain of said DISC1 partner.

8. The method according to any one of claims 1 to 6 wherein said fragment comprises the sequence identified as comprising the DISC1 interacting sequence as shown in Figures 1 C, 2C, 3E or 4G.

9. The method according to any preceding claim wherein the method of detecting an interaction between the DISC1 partner polypeptide and DISCI polypeptide is selected from a FRET assay, gel retardation assay, co-precipitation assay, double fluorescence/cell staining protocol, GST pull-down or other affinity purification method, or a yeast two-hybrid system.

10. An assay according to any one of claims 1 to 9 wherein the respective DISC1 partner polypeptide is encoded by a nucleotide sequence which is capable of hybridising under highly stringent conditions to a sequence as shown in Figures 1A, 2A, 3A, 3C, 4A, 4C, 4E, 5A, 5C or 5E, or fragment thereof.

11. An assay according to any one of claims 1 to 9 wherein the respective DISC1 partner polypeptide is encoded by a nucleotide sequence which is between 70% and 100% identical with a sequence as shown in Figures 1A, 2A, 3A, 3C, 4A, 4C, 4E, 5A, 5C or 5E, or fragment thereof.

12. An expression cassette comprising a promoter operably linked to a polynucleotide sequence as substantially shown in Figures 1A, 2A, 3A, 3C, 4A, 4C, 4E, 5A, 5C or 5E or fragment thereof, capable of encoding a DISC1 partner polypeptide, functionally active variant thereof, or mutant variant thereof capable of binding to DISC1 , or a fragment thereof which is capable of binding to DISC1 , for use in an assay according to any preceding claim.

13. Use of a mutant form of ATF4, KIAA1167, KIAA0380, AKAP450 or NUDE1 to identify agents which substantially restore a wild-type functional activity of a native or mutated DISC1 polypeptidde or a mutated form of ATF4, KIAA1167, KIAA0380, AKAP450 or NUDEI .

14. Use of a cell line comprising a polynucleotide fragment comprising a polynucleotide sequence of ATF4, KIAA1167, KIAA0380, AKAP450 or NUDE1 or a fragment, derivative, or orthologue thereof, for identifying a candidate agent which may be useful in treating, ameliorating or preventing schizophrenia, another psychotic disorder or mood disorder.

15. A polynucleotide sequence comprising a iransci.μuoπai reguiaiuiy btjquciiuc and a sequence under the transcriptional control thereof which includes an RNA sequence characterised in that the RNA sequence is anti-sense to an mRNA sequence capable of encoding ATF4, KIAA1167, KIAA0380, AKAP450 or NUDE1 or fragment thereof for use in preventing or substantially inhibiting the synthesis of a functional ATF4, KIAA1167, KIAA0380, AKAP450 or NUDE1.

16. A transgenic animal in which an orthologue to ATF4, KIAA1167, KIAA0380, AKAP450 or NUDE1 has been mutated or deleted in order to reduce or substantially eliminate expression of said orthologue, for use as a model of schizophrenia, other psychotic disorders or mood disorders.

17. In a further aspect, the present invention provides a kit for identifying a candidate agent which modulates an interaction between a DISC1 partner polypeptide and DISC1 polypeptide, said kit comprising:

18. The kit according to claim 17 further comprising detection means for detecting any modulation in the binding of said DISC1 partner to said D1SC1 polypeptide.

19. A method of screening for a candidate agent which modulates the interaction between a DISC1 partner polypeptide and DISC1 polypeptide, said method comprising: a) introducing into a suitable yeast host cell an expression vector comprising a polynucleotide fragment encoding DISC1 , or a fragment, derivative or homologue thereof, fused to the DNA binding domain of the yeast transcription factor GAL4; b) introducing into the same yeast host cell an expression vector comprising a polynucleotide fragment encoding any one of ATF4, KIAA1167, KIAA0380, AKAP450 or NUDE1 , or a fragment, derivative, mutant or homologue thereof, fused to the activation domain of the yeast transcription factor GAL4; c) bringing the host cell from step b) into contact with potential modulatory agents, by growth on medium containing said compounds and peptides; and d) determining whether the agent has influenced the interaction by means of measuring the degree of reporter gene activation.

20. The present invention also provides a method of screening for a candidate agent which modulates an interaction between a DISC1 partner polypeptide and DISC1 polypeptide, said method comprising: a) coupling in vitro transcription/ translation of a polynucleotide fragment encoding DISC1 , or a fragment, derivative or homologue thereof, fused to glutathione-S-transferase (GST); b) incubating the GST-DISC1 fusion from step a) with glutathione-agarase beads to allow binding to occur; c) coupling in vitro transcription/ translation of a polynucleotide fragment encoding any one of ATF4, KIAA1167, KIAA0380, AKAP450 or NUDE1 , or a fragment, derivative, mutant or homologue thereof, in the presence of [³⁵S] methionine; d) mixing the GST-DISC1 fusion bound to agarose beads and the radiolabelled DISC1 partner in the presence of potential modulatory agents; e) isolating agarose beads and associated polypeptides from the reaction; and f) determining whether the agent has influenced the interaction by analysis of the degree of association of radiolabelled DISC1 partner with the agarose beads.

21. A method of diagnosing schizophrenia, other psychotic disorders or mood disorder in an individual, or the potential for an individual to develop such a disorder, said method comprising detecting any change in a polynucleotide or polypeptide sequence encoding ATF4, KIAA1167, KIAA0380, AKAP450 or NUDE1.

22. Use of a polynucleotide sequence encoding ATF4, KIAA1167, KIAA0380, AKAP450 or NUDE1 or a fragment, derivative, mutant or orthologue thereof or sequences complementary to said polynucleotide sequences for use in determining a loss of expression of any one of the DISC1 partners.

23. Use of a polynucleotide fragment comprising a polynucleotide sequence of any one of ATF4, KIAA1167, KIAA0380, AKAP450 or NUDE1 , or a fragment, derivative, or orthblogue thereof, in the manufacture of a medicament for the treatment of schizophrenia, other psychotic disorders and mood disorders.

24. Use of a polypeptide comprising an amino acid sequence of any one of ATF4, KIAA1167, KIAA0380, AKAP450 or NUDE1, or a functionally active fragment, derivative, or orthologue thereof, in the manufacture of a medicament for the treatment of schizophrenia, other psychotic disorders and mood disorders.

25. A pharmaceutical formulation comprising a polynucleotide fragment comprising a polynucleotide sequence of any one of ATF4, KIAA1167, KIAA0380, AKAP450 or NUDE1 , or a fragment, derivative, or orthologue thereof, and a pharmacologically acceptable carrier.

26. A pharmaceutical formulation comprising a polypeptide comprising an amino acid sequence of any one of ATF4, KIAA1167, KIAA0380, AKAP450 or NUDE1 , or a functionally active fragment, derivative, or orthologue thereof, and a pharmacologically acceptable carrier.

27. A kit for detecting one or more mutations in a gene sequence encoding a DISC1 partner selected from the group consisting of ATF4, KIAA1167, KIAA0380, AKAP450 and NUDE1 , said kit comprising:

(a) oligonucleotides capable of binding to the gene of a DISC1 partner; and

(b) necessary reagents.

28. An antibody specifically reactive against an epitope or epitopes of ATF4, KIAA1167, KIAA0380, AKAP450 and NUDE1 for use in an assay according to any one of claims 1 to 11 , or in therapy.

29. A method of screening for and evaluation an agent which affects the expression of ATF4, KIAA1167, KIAA0380, AKAP450 and NUDE1 gene said method comprising administering the agent to a cell line or animal wherein the levels of expression of said gene has been altered, and detecting the effect of said agent on the expression of said gene.