WO2002026936A2 - Expression genetique dans le systeme nerveux central regule par des neuroleptiques - Google Patents

Expression genetique dans le systeme nerveux central regule par des neuroleptiques Download PDF

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WO2002026936A2
WO2002026936A2 PCT/US2001/030695 US0130695W WO0226936A2 WO 2002026936 A2 WO2002026936 A2 WO 2002026936A2 US 0130695 W US0130695 W US 0130695W WO 0226936 A2 WO0226936 A2 WO 0226936A2
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seq
polypeptide
expression
apod
clz
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PCT/US2001/030695
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WO2002026936A9 (fr
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Elizabeth A. Thomas
J. Gregor Sutcliffe
Thomas M. Pribyl
Brian S. Hilbush
Karl W. Hasel
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Digital Gene Technologies, Inc.
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Priority to AU2001296451A priority Critical patent/AU2001296451A1/en
Priority to AU2001296451A priority patent/AU2001296451A8/en
Priority to US10/381,957 priority patent/US20070010664A1/en
Publication of WO2002026936A2 publication Critical patent/WO2002026936A2/fr
Publication of WO2002026936A9 publication Critical patent/WO2002026936A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • Neuropsychiatric disorders including schizophrenia, affective and behavioral disorders, are a heterogeneous group of devastating illnesses that can impair all aspects of a patient's life. Although positive symptoms, such as hallucinations and delusions are often emphasized, the negative symptoms of these disorders prevent patients from functioning in society, maintaining a job or exhibiting proper social behavior.
  • Mental disorders such as schizophrenia, represent a major public health problem that affects not only the patients and families, but imposes a costly impact on the health system and economy as well (Wasylenki, D. A., Can. J. Psych., 39:S35 (1994); Miller, D. D., Pharmacotherapy, 16: 2 (1996)).
  • midbrain dopamine neurons play an important role in the development of neuropathology.
  • many psychiatric disorders are associated with overactive dopaminergic activity in the meso-striatal dopamine system which refers to both the nigro-striatal dopamine pathway (neurons linking the substantia nigra to the striatum), and the meso-limbic dopamine pathway (neurons linking the ventral tegmental area to limbic regions, such as amygdala, olfactory tubercle and the nucleus accumbens, which is often considered a ventral extension of the striatum).
  • Parkinson's Disease is caused by the degeneration of dopamine neurons of the nigro-striatal pathway.
  • neuroleptic drugs that are widely used in the long-term treatment of various psychiatric disorders, such as schizophrenia, include haloperidol and clozapine.
  • the antipsychotic effects of neuroleptic drugs are generally attributed to blockade of D 2 receptors in the meso-limbic dopamine system (Metzler et al., Schizophrenia Bull, 2, 19-76 (1976)).
  • the best evidence for this comes from the excellent correlation observed between the therapeutic potency of neuroleptics and their affinity for binding to the D 2 receptor (Seeman et al., Curr. Opn. Neurol.
  • dopamine antagonists on the brain are well known and include such effects as an increase in dopamine synthesis and catabolism, an increase in the firing rate of dopamine neurons resulting from the inhibition of pre-synaptic dopamine autoreceptors (Grace et al., J. Pharm. Exp. Ther., 238, 1092-1100 (1986), and a potentiation of cyclic AMP formation resulting from the blockade of post-synaptic dopamine receptors (Rupniak et al, Psychopharm., 84, 519-521 (1984)).
  • neuroleptics can cause a series of mild to severe side effects. Some of these side-effects result from the non-specific nature of neuroleptic drugs, including hypotension and tachycardia, which results from alpha-adrenergic receptor blockade, and dry mouth and blurred vision, which results from the blockade of muscarinic acetylcholine receptors.
  • hypotension and tachycardia which results from alpha-adrenergic receptor blockade
  • dry mouth and blurred vision which results from the blockade of muscarinic acetylcholine receptors.
  • extrapyramidal side effects Marsden et al.
  • Extrapyramidal side effects are associated with the blockade of dopamine receptors in the dorsal striatum (Moore et al., Clin. Neuropharmacol, 12, 167-184 (1989) and include such motor deficits as dystonias (muscle spasms), akathisias (motor restlessness), Parkinson's-like symptoms and Tardive Dyskinesia. Roughly 20% of patients taking antipsychotics demonstrate Parkinson's-like symptoms, the blockade of dopamine D 2 receptors in the striatum being functionally equivalent to the degeneration of nigro-striatal dopamine neurons seen in Parkinson's Disease.
  • Tardive Dyskinesia is a syndrome of abnormal involuntary movements that afflicts roughly 25% of patients on neuroleptic treatment (Jeste et al., Psychopharmacol, 106, 154-160 (1992); Casey, D. E., Schizo. Res., 35: S61 (1999)).
  • the danger of this side effect is that it can be potentially irreversible, that is, patients can still have symptoms of Tardive Dyskinesia long after the antipsychotic has been discontinued. This implicates an epigenetic component to the effects of chronic neuroleptic treatment.
  • clozapine differs from haloperidol in its pharmalogical profile, the specific mechanism leading to the lack of motor side effects is unclear.
  • clozapine Since clozapine has high affinity for other neurotransmitter receptors, such as muscarinic, adrenergic and serotonin receptors, it is possible that the antipsychotic actions of clozapine are partly due to blockade of these other receptors, which may restore proper balance of the dopamine input and output pathways of the basal ganglia.
  • neuroleptic drugs Despite the immediate occupancy of dopamine receptors, neuroleptic drugs have a delayed onset of clinical action, which often can be up to several weeks. Further, as discussed above, neuroleptic drugs are characterized by their ability to cause late and long-lasting motor deficits (Marsden et al., Psychol. Med., 10: 55
  • IEGPs immediate early gene proteins
  • haloperidol a typical neuroleptic, induces the expression c-Fos in the rat striatum and nucleus accumbens
  • clozapine an atypical neuroleptic, induces c-Fos in the nucleus accumbens only (Nguyen et al., Proc. Natl. Acad. Sci. (1992); MacGibbon et al., Mol. Brain Res. ("1994); Robertson et al, Neurosci. (1992)).
  • Haloperidol has also been shown to induce expression of other IEGPs, such as FosB, JunB, JunD and Krox24, in the striatum and nucleus accumbens (Rogue et al., Brain Res. Bull 29, 469-472 (1992); Marsden et al, Psych. Med. (1980); Moore et al., Clin. Neuropharmacol (1989)).
  • clozapine has been shown to induce Krox24 and JunB in the nucleus accumbens only (Nguyen et al. (1992); MacGibbon et al. (1994)).
  • NMDA N-methyl-D-aspartate
  • the TOGA TM . method is an improved method for the simultaneous sequence-specific identification of mRNAs in an mRNA population which allows the visualization of nearly every mRNA expressed by a tissue as a distinct band on a gel whose intensity corresponds roughly to the concentration of the mRNA.
  • the method can identify changes in expression of mRNA induced by typical neuroleptics and the expression induced by atypical neuroleptics.
  • the present invention provides polynucleotides and the encoded polypeptides that are regulated by neuroleptic use.
  • the present invention also provides different uses of these polynucleotides and polypeptides.
  • the invention was made while performing studies using the PCR-based Total Gene Expression Analysis (TOGATM) method to analyze the expression patterns of thousands of genes and comparing the expression patterns among time courses following clozapine drug treatment. Genes regulated by clozapine treatment were examined in haloperidol-treated animals for a comparative analysis. TOGATM analysis identified several genes that were altered in their expression in response to clozapine and/or haloperidol administration in mouse brain.
  • TOGATM Total Gene Expression Analysis
  • the TOGA system was used to examine how gene expression in the striatum and nucleus accumbens is regulated by an atypical neuroleptic agent, such as clozapine.
  • an atypical neuroleptic agent such as clozapine.
  • the studies also examined the pattern of expression of neuroleptic-regulated genes in various regions of the brain. Among other things, these studies were used to determine the genes specifically associated with anti-psychotic activity versus those associated with extrapyramidal side effects, which information advances the development of improved antipsychotic therapies.
  • the identified neuroleptic- regulated molecules are useful in therapeutic and diagnostic applications in the treatment of various psychiatric disorders, such as psychoses and addiction-related behavior. Such molecules are also useful as probes as described by their size, partial nucleotide sequence and characteristic regulation pattern associated with neuroleptic administration.
  • the present invention relates to vectors, host cells, antibodies, and recombinant methods for producing the polynucleotides and the polypeptides.
  • One embodiment of the invention provides an isolated nucleic acid molecule comprising a polynucleotide chosen from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:ll, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID N0:15, SEQ ID N0:16, SEQ ID N0:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID N0.31, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO: 38, SEQ ID NO:39
  • an isolated nucleic acid molecule comprising a polynucleotide at least 95% identical to any one of these isolated nucleic acid molecules and an isolated nucleic acid molecule at least ten bases in length that is hybridizable to any one of these isolated nucleic acid molecules under stringent conditions. Any one of these isolated nucleic acid molecules can comprise sequential nucleotide deletions from either the 5 '-terminus or the 3 '-terminus. Further provided is a recombinant vector comprising any one of these isolated nucleic acid molecules and a recombinant host cell comprising any one of these isolated nucleic acid molecules. Also provided is the gene corresponding to the cDNA sequence of any one of these isolated nucleic acids.
  • Another embodiment of the invention provides an isolated polypeptide encoded by a polynucleotide chosen from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO.ll, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO: 38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, S
  • nucleic acid molecule encoding any of these polypeptides, an isolated nucleic acid molecule encoding a fragment of any of these polypeptides, an isolated nucleic acid molecule encoding a polypeptide epitope of any of these polypeptides, and an isolated nucleic acid encoding a species homologue of any of these polypeptides.
  • any one of these polypeptides has biological activity.
  • any one of the isolated polypeptides comprises sequential amino acid deletions from either the C-terminus or the N-terminus.
  • a recombinant host cell that expresses any one of these isolated polypeptides.
  • Yet another embodiment of the invention comprises an isolated antibody that binds specifically to an isolated polypeptide encoded by a polynucleotide chosen from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO.17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO: 38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, S
  • the isolated antibody can be a monoclonal antibody or a polyclonal antibody.
  • Another embodiment of the invention provides a method for preventing, treating, modulating, or ameliorating a medical condition, such as a neuropsychiatric disorder, comprising administering to a mammalian subject a therapeutically effective amount of a polypeptide of the invention or a polynucleotide of the invention.
  • a method for preventing, treating, moduclating or ameliorating schizophrenia is provided.
  • a method for preventing, treating, modulating or ameliorating bipolar disorder is provided.
  • a further embodiment of the invention provides an isolated antibody that binds specifically to the isolated polypeptide of the invention.
  • a preferred embodiment of the invention provides a method for preventing, treating, modulating, or ameliorating a medical condition, such as a neuropsychiatric disorder, comprising administering to a mammalian subject a therapeutically effective amount of the antibody.
  • a method for preventing, treating, moduclating or ameliorating schizophrenia is provided.
  • a method for preventing, treating, modulating or ameliorating bipolar disorders is provided.
  • An additional embodiment of the invention provides a method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject.
  • the method comprises determining the presence or absence of a mutation in a polynucleotide of the invention.
  • a pathological condition or a susceptibility to a pathological condition such as a neuropsychiatric disorder, is diagnosed based on the presence or absence of the mutation.
  • a method for diagnosing schizophrenia is provided.
  • a method for diagnosing bipolar disorders is provided.
  • Yet another embodiment of the invention provides a method of diagnosing a pathological condition or a susceptibility to a pathological condition, such as a neuropsychiatric disorder, in a subject.
  • a pathological condition such as a neuropsychiatric disorder
  • Especially preferred embodiments include methods of diagnosing schizophrenia and bipolar disorders.
  • the method comprises detecting an alteration in expression of a polypeptide encoded by the polynucleotide of the invention, wherein the presence of an alteration in expression of the polypeptide is indicative of the pathological condition or susceptibility to the pathological condition.
  • the alteration in expression can be an increase in the amount of expression or a decrease in the amount of expression.
  • a first biological sample is obtained from a patient suspected of having a neuropsychiatric disorder, for example, schizophrenia or a bipolar disorder, and a second sample from a suitable comparable control source is obtained.
  • the amount of at least one polypeptide encoded by a polynucleotide of the invention is determined in the first and second sample.
  • the amount of the polypeptide in the first and second samples is determined.
  • a patient is diagnosed as having a neuropsychiatric disorder if the amount of the polypeptide in the first sample is greater than or less than the amount of the polypeptide in the second sample.
  • Another embodiment of the invention provides a method for identifying a binding partner to a polypeptide of the invention.
  • a polypeptide of the invention is contacted with a binding partner and it is determined whether the binding partner effects an activity of the polypeptide.
  • Yet another embodiment of the invention is a method of identifying an activity of an expressed polypeptide in a biological assay.
  • a polypeptide of the invention is expressed in a cell and isolated.
  • the expressed polypeptide is tested for an activity in a biological assay and the activity of the expressed polypeptide is identified based on the test results.
  • Still another embodiment of the invention provides a substantially pure isolated DNA molecule suitable for use as a probe for genes regulated in neuropsychiatric disorders, chosen from the group consisting of the DNA molecules shown in SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO: 38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID
  • kits for detecting the presence of a polypeptide of the invention in a mammalian tissue sample comprises a first antibody which immunoreacts with a mammalian protein encoded by a gene corresponding to the polynucleotide of the invention or with a polypeptide encoded by the polynucleotide in an amount sufficient for at least one assay and suitable packaging material.
  • the kit can further comprise a second antibody that binds to the first antibody.
  • the second antibody can be labeled with enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, phosphorescent compounds, or bioluminescent compounds.
  • Another embodiment of the invention provides a kit for detecting the presence of genes encoding a protein comprising a polynucleotide of the invention, or fragment thereof having at least 10 contiguous bases, in an amount sufficient for at least one assay, and suitable packaging material.
  • Yet another embodiment of the invention provides a method for detecting the presence of a nucleic acid encoding a protein in a mammalian tissue sample.
  • a polynucleotide of the invention or fragment thereof having at least 10 contiguous bases is hybridized with the nucleic acid of the sample. The presence of the hybridization product is detected.
  • Figure 1 is a graphical representation of the results of TOGATM runs using a 5'
  • PCR primer with parsing bases CTAA SEQ ID NO: 66
  • the universal 3' PCR primer SEQ ID NO:23
  • the horizontal axis represents the number of base pairs of the molecules in these samples and the vertical axis represents the fluorescence measurement in the TOGA 7 analysis (which corresponds to the relative expression of the molecule of that address).
  • the results of the TOGA runs have been normalized using the methods described in pending U.S. Patent Application Serial No. 09/318,699/U.S., and pending PCT Application Serial No. PCT/USOO/14159, both entitled Methods and System for Amplitude Normalization and Selection of Data Peaks (Dennis Grace, Jayson Durham); and pending U.S. Patent Application Serial No. 09/318,679/U.S. and pending PCT Application Serial No.
  • Figure 2 presents a graphical example of the results obtained when a DST is verified by the Extended TOGA " method using a primer generated from a cloned product (as described below).
  • the PCR product corresponding to SEQ ID NO:37 (CLZ_43) was cloned and a 5' PCR primer was built from the cloned DST (SEQ ID NO:94).
  • the product obtained from PCR with this primer (SEQ ID NO:72) and the universal 3 ' PCR primer (SEQ ID NO:23) (as shown in the top panel) was compared to the length of the original PCR product that was produced in the TOGA reaction with mRNA extracted from the striatum/nucleus accumbens of mice treated with 7.5 mg/kg of clozapine for 12 days using a 5' PCR primer with parsing bases CTAA (SEQ ID NO:66) and the universal 3' PCR primer (SEQ ID NO:23) (as shown in the middle panel).
  • CTAA SEQ ID NO:66
  • SEQ ID NO:23 universal 3' PCR primer
  • Figure 3 A-D compares the results from Real Time PCR validation (A) (as described below) to the TOGA result from three different experiments: the original clozapine experiment (B), a repeated clozapine experiment performed in duplicate (C), and a haloperidol experiment in duplicate (D).
  • TOGA TM and Real Time PCR show that the DST CLZ_43 (SEQ ID NO:37) increases in expression in clozapine treated mice, while is not responsive to haloperidol treatment.
  • Table 5 lists the 5' and 3' primers used in these Real-Time PCR studies.
  • Figure 4A-F is an in situ hybridization analysis using an antisense cRNA probe directed against the 3 'end of CLZ_43 (SEQ ID NO:37) in saline, clozapine, or haloperidol treated mice.
  • Figure 4A-F demonstrates the pattern of CLZ_43 mRNA expression in coronal sections where A, B and C were sectioned at the level of the striatum (containing nucleus accumbens, Nacc, caudateputamen, Cpu, and neocortex, NC) and D, E, and F were sectioned at the level of the thalamus (Thai), hippocampus (Hipp), and hypothalamus (Hyp).
  • Figure 5A-D compares the results from Real Time PCR validation (A) (as described below) to the TOGA result from three different experiments: the original clozapine experiment (B), a repeated clozapine experiment performed in duplicate (C), and a haloperidol experiment in duplicate (D).
  • the Real Time PCR shows that the mouse sequence homolog to human KIAA1451 (SEQ ID NO: 101) increases in expression in both clozapine (2.09-fold) and haloperidol (2.57-fold) treated mice.
  • Figure 6 is a graphical representation of the results of TOGA analysis, similar to Figure 1, using a 5' PCR primer with parsing bases TTGT (SEQ ID NO: 26) and the universal 3' primer (SEQ ID NO: 23), showing PCR products produced from mRNA extracted from the striatum nucleus accumbens of mice treated with 7.5 mg/kg clozapine as follows: control (no clozapine) (Panel A), 45 minutes (Panel B), 7 hours (Panel C), 5 days (Panel D), 12 days (Panel E), and 14 days (Panel F), where the vertical index line indicates a PCR product of about 266 b.p.
  • the vertical line drawn through the five panels represents the DST molecule identified as CLZ_40 (SEQ ID NO: 12).
  • Figure 7 is a graphical representation of the results of TOGA TM analysis using a 5' PCR primer with parsing bases TTGT (SEQ ID NO: 26) and the universal 3' primer (SEQ ID NO: 23), showing PCR products produced from mRNA extracted from the brain of mo ⁇ hine-treated mice as follows: control striatum (PS) (Panel A), acutely treated striatum (AS) (Panel B), withdrawal striatum (WS) (Panel C), control amygdala (PA) (Panel D), acutely treated amygdala (AA) (Panel E), chronically treated amygdala (TA) (Panel F), and withdrawal amygdala (WA) (Panel G), where the vertical index line indicates a PCR product of about 266 b.p. that is more abundant in control striatum than control amygdala and is differentially regulated by mo ⁇ hine in striatum
  • Figure 8 shows a Northern Blot analysis of DST CLZ_40 (TTGT 266) (SEQ ID NO: 12), where an agarose gel containing poly A enriched mRNA from the striatum/nucleus accumbens of clozapine-treated mice as well as size standards was blotted after electrophoresis and probed with radiolabelled CLZ_40.
  • Mice were treated with clozapine (7.5 mg/kg) for the following time durations before mRNA extraction: control (no clozapine), 45 minutes, 7 hours, 5 days, 12 days, and 14 days.
  • Figure 9 is a graphical representation comparing the results of the TOGA T analysis of clone CLZ_40 (SEQ ID NO: 12) shown in Fig. 6 and the Northern Blot analysis of clone CLZ_40 shown in Figure 8.
  • Figure 10A-D compares the results from Real Time PCR validation (A) (as described below) to the TOGATM result from three different experiments: the original clozapine experiment (B), a repeated clozapine experiment performed in duplicate (C), and a haloperidol experiment in duplicate (D).
  • A Real Time PCR validation
  • B original clozapine experiment
  • C repeated clozapine experiment performed in duplicate
  • D haloperidol experiment in duplicate
  • Table 5 lists the 5' and 3' primers used in these Real-Time PCR studies.
  • Figure 11A-B is an in situ hybridization analysis, showing DST CLZ_40 (SEQ
  • ID NO: 12 mRNA expression in mouse brain using an antisense cRNA probe directed against the 3' end of CLZ_40, where 15 A shows expression in the nucleus accumbens (Acb) and pyriform cortex (Pir) and 15B shows expression in the dentate gyrus (DG).
  • Figure 12 is a graphical representation of the results of TOGA analysis using a 5' PCR primer with parsing bases CACC (SEQ ID NO: 25) and universal 3' primer (SEQ ID NO: 23), showing PCR products produced from mRNA extracted from the striatum/nucleus accumbens of mice treated with 7.5 mg/kg of clozapine for the following durations: control (no clozapine) (Panel A), 45 minutes (Panel B), 7 hours (Panel C), 5 days (Panel D), 12 days (Panel E), and 14 days (Panel F), where the vertical index line indicates a PCR product of about 201 b.p. that is present in the control sample and increasingly enriched over time in the clozapine-treated samples.
  • the vertical line drawn through the five panels represents the DST molecule identified as CLZ_5 (SEQ ID NO:2).
  • Figure 13 shows a Northern Blot analysis of clone CLZ_5 (CACC 201) (SEQ ID NO: 1
  • Figure 14 is a graphical representation comparing the results of the TOGATM analysis of DST CLZ_5 (SEQ ID NO: 2) shown in Fig. 12 and the Northern Blot analysis of clone CLZ_5 shown in Figure 13.
  • Figure 15A-D compares the results from Real Time PCR validation (A) (as described below) to the TOGA T result from three different experiments: the original clozapine experiment (B), a repeated clozapine experiment performed in duplicate (C), and a haloperidol experiment in duplicate (D).
  • A Real Time PCR validation
  • B original clozapine experiment
  • C repeated clozapine experiment performed in duplicate
  • D haloperidol experiment in duplicate
  • Table 5 lists the 5' and 3' primers used in these Real-Time PCR studies.
  • Figure 16A-C is an in situ hybridization analysis using an antisense cRNA probe directed against the 3' end of CLZ_5 (SEQ ID NO:2), showing the pattern of CLZ_5 mRNA expression in mouse anterior brain (16A), midbrain (16B), and posterior brain (16C), where CLZ_5 is expressed in scattered glial cells and white matter tracts.
  • Figure 17A-I is an in situ hybridization analyses, using an antisense cRNA probe directed against the 3' end of CLZ_5 (SEQ ID NO:2), showing CLZ_5 mRNA expression in mouse anterior brain (17A-C), midbrain (17D-F), and posterior brain (17G-I) in saline-treated mice (top row), mice treated with clozapine for 5 days (middle row), and mice treated with clozapine for 14 days (bottom row), where the clozapine treatment induces expression in the glial cells.
  • Figure 18A-H shows a darkfield photomicrograph of various brain regions, including the co ⁇ us callosum (cc, Fig. 189A, E); caudate putamen (CPu, Fig. 18B, F); anterior commissure (aca, Fig. 18C, G); and globus pallidus (GP, Fig. 18D, H) in control (18A-D) and clozapine-treated (18E-H) animals.
  • the figure demonstrates upregulated ApoD mRNA expression in various brain regions.
  • Figure 19A-D shows a darkfield photomicrograph in the internal capsule (ic) (19A, B) and a brightfield view of the optic tract (opt) (19C, D) from control (19A, C) and clozapine-treated (19B, D) animals.
  • the figure demonstrates up-regulated apoD mRNA expression in the internal capsule (ic).
  • Figure 20A-F shows GFAP and apoD co-localization in the striatum (20A, B,
  • Figure 21 shows a Northern Blot analysis of clone CLZ_5 (SEQ ID NO: 2), where an agarose gel containing poly A enriched mRNA from cultured glial cells treated with clozapine as well as size standards was blotted after electrophoresis and probed with radiolabelled CLZ_5.
  • Figure 22A-B are Western blot analyses showing the distribution of apoD expression in human brain, wherein Western blots containing 50 ⁇ g total protein/lane were probed with a monoclonal antibody directed against human apoD and enhanced chemiluminescence (ECL) was used to detect immunoreactivity.
  • Figure 22A was visualized by 30 second exposure to autoradiography film and Figure 22B was visualized by 90 second exposure to autoradiography film.
  • Figure 23 A and B shows Western blot analyses (23A) and densitrometric data (23B) demonstrating apoD expression in dorsolateral prefrontal cortex of eight control (Con-1 to Con-8) and eight schizophrenic subjects (Sch-1 to Sch-8), wherein 50 ⁇ g total protein/lane were probed with a monoclonal antibody directed against human ApoD and enhanced chemiluminescence (ECL) was used to detect immunoreactivity.
  • ECL enhanced chemiluminescence
  • Figure 26A-B are scatter plots showing ApoD levels in the serum of male versus female subjects (26A) and subjects ranging in age from 20-65 (26B).
  • Figure 27 shows regional ApoD gene expression in the brain was compared in young nontransgenic (Yg-NT), young transgenic (Yg-TG), aged nontransgenic (Aged-N) and aged transgenic (Aged-TG) mice.
  • Yg-NT young nontransgenic
  • Yg-TG young transgenic
  • Aged-N aged nontransgenic
  • Aged-TG aged transgenic
  • apoD expression in young PDAPP Tg mice did not differ significantly from young wild type mice.
  • apoD increased significantly as compared to young mice.
  • These increases in expression were most notable in the white matter tracts: hippocampal fimbria, co ⁇ us callosum, septal white matter tracts.
  • Comparison between aged wild type and PDAPP mice revealed that the PDAPP mice had greater apoD expression as compared to the wild type. Representative sections were taken at four different levels.
  • Panel A taken at the level of the caudatoputamen (CP) demonstrating gene expression in the co ⁇ us callosum (cc) and septal white matter tracts (sp).
  • Panel B taken at the level of the globus pallidus (GP) demonstrating gene expression in the hippocampal fimbria (fi) and co ⁇ us callosum (cc).
  • Panel C, D at the level of the hippocampus (Hipp) and thalamus (Th) demonstrating gene expression in the co ⁇ us callosum (cc).
  • Figure 28 shows at the cellular level, the number of individual glial cells that express apoD in the co ⁇ us callosum (A) and hippocampal fimbria (B) of the PDAPP mice as compared to the wild type mice was significant.
  • apoD gene expression is increased moderately in the young transgenic mice (Y-Tg) as compared to the young nontransgenic mice (Y- NT).
  • ApoD mRNA expression in the medial co ⁇ us callosum is significantly increased in the aged transgenic (Aged-Tg) as compared to the aged nontransgenic (Aged-NT).
  • Hipp-fi hippocampal fimbria
  • apoD gene expression is increased significantly in the Aged Tg as compared to Aged-NT.
  • Figure 29 shows the increase in ApoD expression at the cellular level in the co ⁇ us callosum and hippocampal fimbria were quantified by determining the number of cells expressing apoD mRNA within a defined field of view.
  • Cell counts were performed using a 20X objective in both brightfield and darkfield on 4 different slices from each of the 2 regions, the co ⁇ us callosum (A) and the hippocampal fimbria (B).
  • a total of 4 animals from each group was analyzed (young nontransgenic, Yg-NT; young transgenic, Yg-Tg; aged transgenic, Aged-NT; aged nontransgenic, Aged-Tg).
  • Figure 30A-B is an in situ hybridization analysis using an antisense cRNA probe directed against the 3 ' end of DST CLZ_3 (SEQ ID NO : 1 ), showing the pattern of CLZ_3 mRNA expression in a coronal section through the hemispheres at level of hippocampus (30 A) and cross section through midbrain (30B) in mouse brain.
  • Figure 31 A-B is an in situ hybridization analysis using an antisense cRNA probe directed against the 3' end of CLZ_16 (SEQ ID NO:15), showing the pattern of CLZ_16 mRNA expression in coronal sections through hemispheres in mouse brain.
  • Figure 31A shows dense labeling in the cortex and surrounding the hippocampal formation as well as moderate labeling in the dorsal thalamus and posterior brain.
  • Figure 3 IB shows uniform labeling throughout.
  • Figure 32 shows CLZ_17 (SEQ ID NO: 28) mRNA expression in the brain was determined by in situ hybridization using riboprobes specific to the DST. In (A) CLZ_17 expression was observed in the septal nucleus (SPT).
  • CLZ_17 expression was observed in the hypothalamic nuclei (HYP) and SPT.
  • CLZ_17 was observed in the hippocampus (HIP) and the HYP.
  • CLZ_17 was observed in the amygdala (AMYG), the HYP, and the HIP.
  • Figure 33A-B is an in situ hybridization analysis using an antisense cRNA probe directed against the 3' end of CLZ_24 (SEQ ID NO: 7), showing the pattern of CLZ_24 mRNA expression in a coronal section through the hemispheres (33 A) and cross section through the brainstem (33B) in mouse brain.
  • Figure 34A-B is an in situ hybridization analysis using an antisense cRNA probe directed against the 3' end of CLZ 26 (SEQ ID NO:29), showing the pattern of CLZ_26 mRNA expression in a coronal section of the hemispheres at the level of hippocampal formation (34A) and coronal section of the hemispheres at the level of striatum (34B) in mouse brain.
  • Figure 35A-B is an in situ hybridization analysis using an antisense cRNA probe directed against the 3' end of CLZ_28 (SEQ ID NO:30), showing the pattern of CLZ_28 mRNA expression in a coronal section through the hemispheres at the level of hippocampus (35 A) and coronal section through the posterior region of hemispheres (35B) in mouse brain.
  • Figure 36A-B is an in situ hybridization analysis using an antisense cRNA probe directed against the 3' end of CLZ_34 (SEQ ID NO:9) showing the pattern of CLZ_34 mRNA expression in a coronal section through the hemispheres at the level of hippocampus (36 A) and cross section through the midbrain (36B) in mouse brain.
  • Figure 37 is a graphical representation of a Northern Blot analysis of clone CLZ_38 (TGCA 109) (SEQ ID NO: 11), where an agarose gel containing poly A enriched mRNA from the striatum/nucleus accumbens of clozapine-treated mice as well as size standards was blotted after electrophoresis and probed with radiolabelled CLZ_38. Mice were treated with clozapine (7.5 mg/kg) for the following time durations before mRNA extraction: control (no clozapine), 45 minutes, 7 hours, 5 days, 12 days, and 14 days.
  • Figure 38 shows CLZ_38 mRNA expression in the brain was determined by in situ hybridization using riboprobes specific to the DST.
  • CLZ_38 expression was observed primarily in the white matter tracts of the brain.
  • CLZ_38 is expression is observed in the co ⁇ us callosum (cc) and anterior commisure (ac).
  • cc co ⁇ us callosum
  • ac anterior commisure
  • sp white matter of the septum
  • CLZ_38 is expressed by cells in the hippocampal fimbria (fi).
  • fi hippocampal fimbria
  • D CLZ_38 expression is observed in the cc, fi, and optic tract (opt).
  • Figure 39 is a graphical representation of a Northern Blot analysis of clone CLZ_44 (ACGG 352) (SEQ ID NO:38), where an agarose gel containing poly A enriched mRNA from the striatum/nucleus accumbens of clozapine-treated mice as well as size standards was blotted after electrophoresis and probed with radiolabelled CLZ_44. Mice were treated with clozapine (7.5 mg/kg), haloperidol (4 mg/kg), or ketanserin (4 mg/kg) for two weeks before mRNA extraction.
  • Figure 40A-B is an in situ hybridization analysis using an antisense cRNA probe directed against the 3' end of CLZ_44 (SEQ ID NO: 38), showing the pattern of CLZ_44 mRNA expression in a coronal section showing labeling in the hippocampus, hypothalamus, and temporal cortex (40A) and coronal section showing cortical labeling (40B) in mouse brain.
  • Figure 41 A-B is an in situ hybridization analysis using an antisense cR A probe directed against the 3' end of CLZ_64 (SEQ ID NO:48), showing the pattern of CLZ_64 mRNA expression in different coronal sections of the hemispheres in mouse brain.
  • isolated nucleic acid refers to a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of naturally occurring genomic nucleic acid.
  • the term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid inco ⁇ orated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding
  • isolated polypeptide refers to a polypeptide removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state.
  • isolated antibody refers to an antibody removed from its original environment (e.g., the natural environment ifit is naturally occurring), and thus is altered “by the hand of man” from its natural state.
  • isolated refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state.
  • Polynucleotide or “polynucleotide of the invention” or “polynucleotide of the present invention” refers to a molecule having a nucleic acid sequence contained in SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:ll, SEQ ID NO:12, SEQ ID NO:13 f SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO: 38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQEQ ID
  • the polynucleotide can contain all or part of the nucleotide sequence of the full length cDNA sequence, including the 5' and 3' untranslated sequences, the coding region, with or without the signal sequence, the secreted protein coding region, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence.
  • a polynucleotide of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • a polynucleotide can be composed of triple- stranded regions comprising RNA or DNA or both RNA and DNA.
  • a polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons.
  • Modified bases include, for example, tritylated bases and unusual bases such as inosine.
  • polynucleotide embraces chemically, enzymatically, or metabolically modified forms.
  • a "polynucleotide” of the present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:ll, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO: 38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, S
  • washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5X SSC). Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments.
  • Typical blocking reagents include Denhardt's reagent, BLOTTO (5% w/v non-fat dried milk in phosphate buffered saline ("PBS), heparin, denatured salmon sperm DNA, and other commercially available proprietary formulations.
  • PBS phosphate buffered saline
  • heparin 5% w/v non-fat dried milk in phosphate buffered saline
  • denatured salmon sperm DNA and other commercially available proprietary formulations.
  • the inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
  • polynucleotide which hybridizes only to polyA+ sequences (such as any 3' terminal polyA ⁇ tract of a cDNA shown in the sequence listing), or to a complementary stretch of T (or U) residues, would not be included in the definition of "polynucleotide,” since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone).
  • Polypeptide or “polypeptide of the invention” or “polypeptide of the present invention” refers to a molecule having a translated amino acid sequence generated from the polynucleotide as broadly defined.
  • the translated amino acid sequence beginning with the methionine, is identified although other reading frames can also be easily translated using known molecular biology techniques.
  • the polypeptides produced by the translation of these alternative open reading frames are specifically contemplated by the present invention.
  • the polypeptide of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids.
  • polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. See references below. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching.
  • Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formulation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation
  • a polypeptide has "biological activity" when the polypeptide has structural, regulatory or biochemical functions of a naturally occurring molecule.
  • Biological activity can be measured by several kinds of biological assays, both in vitro (e.g., cell cultures) or in vivo (e.g., behavioral or metabolic assays). In these cases, the potency of the biological activity is measured by its dose-response characteristics; in the case of polypeptides with activity similar to the polypeptide of the present invention, the dose-response dependency will be substantially similar in a given activity as compared to the polypeptide of the present invention.
  • Polypeptides may derive their "biological activity" through binding to specific cellular receptors, which -mediate secondary signals to the target cell or tissue.
  • peptides may interact directly with other proteins or other molecules, and alter their conformation of function, or they may block the binding of a third molecule to the same interaction site, thereby affecting the singal normally mediated between the two molecules.
  • DNA refers to deoxyribonucleic acid.
  • RNA refers to ribonucleic acid
  • mRNA refers to messenger ribonucleic acid.
  • cDNA refers to a deoxyribonucleic acid that is complementary to an mRNA.
  • Gene refers to a region of DNA that controls a discrete hereditary characteristic, usually corresponding to a single protein or RNA. This definition includes the entire functional unit encompassing coding DNA sequence, the regions preceding and following the coding region (leader or trailer), noncoding regulatory DNA sequences, and introns. "Codon” refers to the three-nucleotide sequence of an mRNA molecule that codes for one specific amino acid.
  • Vector refers to a vehicle for transfer of DNA into a recipient cell.
  • Standard mutation or “silent substitution” refers to a mutation that causes no functional change in the gene product.
  • Phenotype refers to the appearance, behavior, or other characteristics of a cell or individual due to actual expression, or pattern of expression, of a specific gene or set of genes. Differences in phenotype may be due to changes in the expression or pattern of expression of a specific gene or set of genes, or to differences in the biological activity of one or more genes. These differences may be a result of polymo ⁇ hic or allelic differences in the coding region of the specific genes or in their regulatory sequences, or to other genetic variations (e.g., new mutations).
  • Hybridization refers to the time- and temperature-dependent process by which two complementary single-stranded polynucleotides associate to form a double helix.
  • Probe refers to a polynucleotide, often radiolabelled, used to detect complementary sequences, e.g. an mRNA used to locate its gene by a corresponding nucleic acid blotting method.
  • Constant amino acid substitution refers to a substitution between similar amino acids that preserves an essential chemical characteristic of the original polypeptide.
  • “Phage” refers to a virus that infects bacteria. Many phage have proved useful in the study of molecular biology and as vectors for the transfer of genetic information between cells. "Plasmid” refers to a self-replicating extra-chromosomal element, usually a small segment of duplex DNA that occurs in some bacteria; used as a vector for the introduction of new genes into bacteria.
  • Retrovirus refers to a virus with an RNA genome that may be either an mRNA, (+)-RNA, or its complement, (-)-RNA.
  • Class 1 contains (+)-RNA; class 2, (- )-RNA, which is the template for an RNA-dependent RNA polymerase; class 3, double-stranded RNA, in which (+)-RNA is synthesized by an RNA-dependent RNA polymerase; class 4, retrovirus, in which (+)-RNA is a template for an RNA- dependent DNA polymerase (a reverse transcriptase).
  • a Retrovirus may be used as a vector for the introduction of genes into mammalian cells.
  • Multiple Helix refers to the tertiary structure of collagen that twists three polypeptide chains around themselves; also a triple-stranded DNA structure that involves Hoogstein base pairing between B-DNA and a third DNA strand that occupies the major groove.
  • Antibody refers to an immunoglobulin molecule that reacts specifically with another (usually foreign) molecule, the antigen.
  • mAb Monoclonal antibody
  • mAb refers to an immunoglobulin preparation that is completely homogeneous, due to its formation by daughters of a single progenitor cell that has been programmed for the synthesis and secretion of one specific antibody.
  • Polyclonal antibody refers to a heterogeneous immunoglobulin preparation that contains antibodies directed against one or more determinants on an antigen; the product of daughters of several progenitor cells that have been programmed for immunoglobulin synthesis and secretion.
  • “Complementary” as used in nucleic acid chemistry is descriptive of the relationship between two polynucleotides that can combine in an antiparallel double helix; the bases of each polynucleotide are in a hydrogen-bonded inter-strand pair with a complementary base, A to T (or U) and C to G.
  • a to T or U
  • C to G.
  • protein chemistry the matching of shape and/or charge of a protein to a ligand.
  • C-terminus refers to, in a polypeptide, the end with a free carboxyl group.
  • N-terminus refers to, in a polypeptide, the end with a free amino group.
  • a “secreted” protein refers to those proteins capable of being directed to the endoplasmic reticulum, secretory vesicles, or the extracellular space as a result of a signal sequence, as well as those proteins released into the extracellular space without necessarily containing a signal sequence. If the secreted protein is released into the extracellular space, the secreted protein can undergo extracellular processing to produce a "mature" protein. Release into the extracellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage.
  • Variant refers to a polynucleotide or polypeptide differing from the polynucleotide or polypeptide of the present invention, but retaining essential properties thereof. In general, variants have close similarity overall and are identical in many regions to the polynucleotide or polypeptide of the present invention.
  • identity is well known to skilled artisans (Carillo et al, SIAMJ Applied Math., 48:1073 (1988)). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in "Guide to Huge Computers,” Martin J. Bishop, Ed., Academic Press, San Diego, (1994) and Carillo et al., (1988), Supra. "Epitopes” refer to polypeptide fragments having antigenic or immunogenic activity in an animal, especially in a human.
  • a preferred embodiment of the present invention relates to a polypeptide fragment comprising an epitope, as well as the polynucleotide encoding this fragment.
  • a region of a protein molecule to which an antibody can bind is defined as an "antigenic epitope.”
  • an antibody to which an antibody can bind.
  • immunological epitope is defined as a part of a protein that elicits an antibody response. (See, e.g., Geysen et al, Proc. Natl. Acad. Sci. USA, 81:3998-4002 (1983)).
  • Homologous means corresponding in structure, position, origin or function.
  • a “homologous polynucleotide” refers to a polynucleotide which encodes a homologous polypeptide.
  • a "homologous nucleic acid molecule” refers to a nucleic acid molecule which encodes a homologous polypeptide.
  • a “homologous polypeptide” refers to a polypeptide having any of the following characteristics with respect to the polypeptides of the present invention: similar function, similar amino acid sequence, similar subunit structure and formation of a functional heteropolymer, immunological cross-reaction, similar expression profile, similar subcellular location, similar substrate specificity, or similar response to specific inhibitors.
  • ELISA refers to an enzyme-linked immunosorbent assay that employs an antibody or antigen bound to a solid phase and an enzyme-antigen or enzyme- antibody conjugate to detect and quantify the amount of an antigen present in a sample.
  • a “specific binding agent” refers to a molecular entity capable of selectively binding a reagent species of the present invention or a complex containing such a species, but is not itself a polypeptide or antibody molecule composition of the present invention.
  • the word "complex” as used herein refers to the product of a specific binding reaction such as an antibody-antigen or receptor-ligand reaction. Exemplary complexes are immunoreaction products.
  • label and "indicating means” in their various grammatical forms refer to single atoms and molecules that are either directly or indirectly involved in the production of a detectable signal to indicate the presence of a complex.
  • package refers to a solid matrix or material such as glass, plastic
  • a package can be a glass vial used to contain milligram quantities of a contemplated polypeptide or antibody or it can be a microtiter plate well to which microgram quantities of a contemplated polypeptide or antibody have been operatively affixed (i.e., linked) so as to be capable of being immunologically bound by an antibody or antigen, respectively.
  • Instructions for use typically include a tangible expression describing the reagent concentration or at least one assay method parameter, such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions, and the like.
  • DST refers to a Digital Sequence Tag, i.e., a polynucleotide that is an expressed sequence tag of the 3 ' end of an mRNA.
  • mice Male C57B1/6J mice (20-28 g) were housed in groups of four on a standard 12/12 hour light-dark cycle with ad libitum access to standard laboratory chow and tap water. For the experimental paradigms, mice were divided into groups of 25 and subjected to the following treatments:
  • Control groups Mice received a single injection of sterile saline (0.1 ml volume), or no injection, and were sacrificed after 45 minutes.
  • Acute neuroleptic treatment Mice received a single intraperitoneal injection of the atypical neuroleptic clozapine (7.5 mg/kg). Animals were sacrificed after 45 minutes.
  • Chronic neuroleptic treatment Mice received daily subcutaneous injections of clozapine (7.5 mg/kg) or haloperidol (4 mg/kg) for time periods of 5 days to 2 weeks.
  • cytoplasmic RNA was isolated by phenol: chloroform extraction of the homogenized tissue according to the method described in Schibler et al, J. Mol. Bio., 142, 93-116 (1980). Poly A enriched mRNA was prepared from cytoplasmic RNA using well- known methods of oligo dT chromatography. Isolated RNA was then analyzed using a method of simultaneous sequence-specific identification of mRNAs known as TOGATM (TOtal Gene expression Analysis) described below.
  • TOGATM TOtal Gene expression Analysis
  • TOGATM TOtal Gene expression Analysis
  • the isolated RNA was enriched to form a starting polyA-containing mRNA population by methods known in the art.
  • the TOGATM method further comprised an additional PCR step performed using four 5' PCR primers in four separate reactions and cDNA templates prepared from a population of antisense cRNAs.
  • a final PCR step that used 256 5' PCR primers in separate reactions produced PCR products that were cDNA fragments that corresponded to the 3 '-region of the starting mRNA population.
  • the produced PCR products were then identified by: a) the initial 5' sequence comprising the sequence remainder of the recognition site of the restriction endonuclease used to cut and isolate the 3' region plus the sequence of the preferably four parsing bases immediately 3' to the remainder of the recognition site, preferably the sequence of the entire fragment, and b) the length of the fragment. These two parameters, sequence and fragment length, were used to compare the obtained PCR products to a database of known polynucleotide sequences. Since the length of the obtained PCR products includes known vector sequences at the 5' and 3' ends of the insert, the sequence of the insert provided in the sequence listing is shorter than the fragment length that forms part of the digital address.
  • the method yields Digital Sequence Tags (DSTs), that is, polynucleotides that are expressed sequence tags of the 3' end of mRNAs. DSTs that showed changes in relative levels during intraperitoneal injection clozapine were selected for further study. The intensities of the laser-induced fluorescence of the labeled PCR products were compared across samples isolated.
  • DSTs Digital Sequence Tags
  • double-stranded cDNA is generated from poly(A)-enriched cytoplasmic RNA extracted from the tissue samples of interest using an equimolar mixture or set of all 48 5 '-biotinylated anchor primers to initiate reverse transcription.
  • One such suitable set is G-A-A-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-G-C-A-G- G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-N-N-N (SEQ ID NO: 20), where V is A, C or G and N is A, C, G or T.
  • One member of this mixture of 48 anchor primers initiates synthesis at a fixed position at the 3' end of all copies of each mRNA species in the sample, thereby defining a 3' endpoint for each species, resulting in biotinylated double-stranded cDNA.
  • Each biotinylated double-stranded cDNA sample was cleaved with the restriction endonuclease Mspl, which recognizes the sequence CCGG.
  • the resulting fragments of cDNA corresponding to the 3' region of the starting mRNA were then isolated by capture of the biotinylated cDNA fragments on a streptavidin-coated substrate.
  • Suitable streptavidin-coated substrates include microtitre plates, PCR tubes, polystyrene beads, paramagnetic polymer beads, and paramagnetic porous glass particles.
  • a preferred streptavidin-coated substrate is a suspension of paramagnetic polymer beads (Dynal, Inc., Great Neck, NY).
  • the cDNA fragment product was released by digestion with Notl, which cleaves at an 8-nucleotide sequence within the anchor primers but rarely within the mRNA-derived portion of the cDNAs.
  • Notl which cleaves at an 8-nucleotide sequence within the anchor primers but rarely within the mRNA-derived portion of the cDNAs.
  • the 3' Mspl-Notl fragments which are of uniform length for each mRNA species, were directionally ligated into Clal- Notl- cleaved plasmid pBC SK+ (Stratagene, La Jolla, CA) in an antisense orientation with respect to the vector's T3 promoter, and the product used to transform Escherichia coli SURE cells (Stratagene).
  • the ligation regenerates the Notl site, but not the Mspl site, leaving CGG as the first 3 bases of the 5' end of all PCR products obtained.
  • Each library contained in excess of 5 x IO 5 recombinants to ensure a high likelihood that the 3' ends of all mRNAs with concentrations of 0.001% or greater were multiply represented.
  • Plasmid preps (Qiagen) were made from the cDNA library of each sample under study. An aliquot of each library was digested with Mspl, which effects linearization by cleavage at several sites within the parent vector while leaving the 3 ' cDNA inserts and their flanking sequences, including the T3 promoter, intact.
  • the product was incubated with T3 RNA polymerase (MEGAscript kit, Ambion) to generate antisense cRNA transcripts of the cloned inserts containing known vector sequences abutting the Mspl and Notl sites from the original cDNAs.
  • T3 RNA polymerase MEGAscript kit, Ambion
  • each of the cRNA preparations was processed in a three-step fashion.
  • 250 ng of cRNA was converted to first-strand cDNA using the 5' RT primer (A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-G, (SEQ ID NO: 21).
  • step two 400 pg of cDNA product was used as PCR template in four separate reactions with each of the four 5' PCR primers of the form G-G-T-C-G-A-C-G-G-T-A-T-C-G- G-N (SEQ ID NO: 22), each paired with a "universal" 3' PCR primer G-A-G-C-T-C- C-A-C-C-G-C-G-G-G-T (SEQ ID NO: 23) to yield four sets of PCR reaction products ("Nl reaction products").
  • step three the product of each subpool was further divided into 64 subsubpools (2ng in 20 ⁇ l) for the second PCR reaction.
  • This PCR reaction comprised adding 100 ng of the fluoresceinated "universal" 3' PCR primer (SEQ ID NO: 23) conjugated to 6-FAM and 100 ng of the appropriate 5' PCR primer of the form C-G- A-C-G-G-T-A-T-C-G-G-N-N-N-N (SEQ ID NO: 24), and using a program that included an annealing step at a temperature X slightly above the Tm of each 5' PCR primer to minimize artifactual mispriming and promote high fidelity copying.
  • Each polymerase chain reaction step was performed in the presence of TaqStart antibody (Clonetech).
  • the products (“N4 reaction products”) from the final polymerase chain reaction step for each of the tissue samples were resolved on a series of denaturing DNA sequencing gels using the automated ABI Prizm 377 sequencer. Data were . collected using the GeneScan software package (ABI) and normalized for amplitude and migration. Complete execution of this series of reactions generated 64 product subpools for each of the four pools established by the 5' PCR primers of the first PCR reaction, for a total of 256 product subpools for the entire 5' PCR primer set of the second PCR reaction.
  • mice treated with 7.5 mg/kg of clozapine for the following durations: control (no clozapine), 45 minutes, 7 hours, 5 days, 12 days, and 14 days were analyzed.
  • Table 1 is a summary of the expression levels of 496 mRNAs determined from cDNA. These cDNA molecules are identified by their digital address, that is, a partial 5' terminus nucleotide sequence coupled with the length of the molecule, as well as the relative amount of the molecule produced at different time intervals after treatment.
  • the 5' terminus partial nucleotide sequence is determined by the recognition site for Mspl (CC GG) and the nucleotide sequence of the parsing bases of the 5' PCR primer used in the final PCR step.
  • the digital address length of the fragment was determined by inte ⁇ olation on a standard curve and, as such, may vary ⁇ 1-2 b.p. from the actual length as determined by sequencing.
  • the entry in Table 1 that describes a DNA molecule identified by the digital address Mspl CTAA 461 is further characterized as having a 5' terminus partial nucleotide sequence of CGGCTAA and a digital address length of 461 b.p.
  • the DNA molecule identified as Mspl CTAA 461 is further described as being expressed at increasing levels after 12 days of treatment with clozapine (see Figure 1). Additionally, the DNA molecule identified as Mspl CTAA 461 is described by its nucleotide sequence, which corresponds with SEQ ID NO: 37.
  • the other DNA molecules identified in Table 1 by their Mspl digital addresses are further characterized by: 1) the level of gene expression in the striatum/nucleus accumbens of mice without clozapine treatment (control), 2) the level of gene expression in the striatum/nucleus accumbens of mice treated with clozapine for 45 minutes, 3) the level of gene expression in the striatum/nucleus accumbens of mice treated with clozapine for 7 hours, 4) the level of gene expression in the striatum/nucleus accumbens of mice treated with clozapine for 5 days, 5) the level of gene expression in the striatum/nucleus accumbens of mice treated with clozapine for 12 days, 6) the level of gene expression in the striatum/nucleus accumbens of mice treated with clozapine for 14 days.
  • DSTs were further characterized as shown in the Tables and their nucleotide sequences are provided as SEQ ID NOs: 1-12; 14-19; 28- 31; and 36-50 in the Sequence Listing below.
  • the ligation of the sequence into a vector does not regenerate the Mspl site; the experimentally determined sequence reported herein has C-G-G as the first bases of the 5' end.
  • Figure 1 The data shown in Figure 1 were generated with a 5' -PCR primer (C-G-A-C- G-G-T-A-T-C-G-G-C-T-A-A; SEQ ID NO: 66) paired with the "universal" 3 ' primer (SEQ ID NO:23) labeled with 6-carboxyfluorescein (6FAM, ABI) at the 5' terminus.
  • PCR reaction products were resolved by gel electrophoresis on 4.5%> acrylamide gels and fluorescence data acquired on ABI377 automated sequencers. Data were analyzed using GeneScan software (Perkin-Elmer).
  • Figure 1 is a graphical representation of the results of TOGATM runs using a 5'
  • PCR primer with parsing bases CTAA SEQ ID NO: 66
  • the universal 3' PCR primer SEQ ID NO:23
  • the horizontal axis represents the number of base pairs of the molecules in these samples and the vertical axis represents the fluorescence measurement in the TOGA T analysis (which corresponds to the relative expression of the molecule of that address).
  • the results of the TOGATM runs have been normalized using the methods described in pending U.S. Patent Application Serial No. 09/318,699/U.S., and pending PCT Application Serial No. PCT/USOO/14159, both entitled Methods and System for Amplitude Normalization and Selection of Data Peaks (Dennis Grace, Jayson Durham); and pending U.S. Patent Application Serial No. 09/318,679/U.S. and pending PCT Application Serial No.
  • the PCR product was isolated, cloned into a TOPO vector
  • the TOGA PCR product was sequenced using a modification of a direct sequencing methodology (Innis et al., Proc. Nat'l. Acad. Set, 85: 9436-9440 (1988)).
  • PCR products corresponding to DSTs were gel purified and PCR amplified again to inco ⁇ orate sequencing primers at 5' and 3 ' ends.
  • the sequence addition was accomplished through 5' and 3' ds-primers containing Ml 3 sequencing primer sequences (Ml 3 forward and M13 reverse respectively) at their 5' ends, followed by a linker sequence and a sequence complementary to the DST ends.
  • a master mix containing all components except the gel purified PCR product template was prepared, which contained sterile H 2 O, 10X PCR II buffer, lOmM dNTP, 25 mM MgCl 2 , AmpliTaq/Antibody mix (1.1 ⁇ g/ ⁇ l Taq antibody, 5 U/ ⁇ l AmpliTaq), 100 ng/ ⁇ l of 5" ds-primer (5' TCC CAG TCA CGA CGT TGT AAA ACG ACG GCT CAT ATG AAT TAG GTG ACC GAC GGT ATC GG 3', SEQ ID NO: 52), and 100 ng/ ⁇ l of 3' ds-primer (5' CAG CGG ATA ACA ATT TCA CAC AGG GAG CTC CAC CGC GGT GGC GGC C 3', SEQ ID NO: 53).
  • PCR was performed using the following program: 94°C, 4 minutes and 25 cycles of 94°C, 20 seconds; 65°C, 20 seconds; 72°C, 20 seconds; and 72°C 4 minutes.
  • the resulting amplified adapted PCR product was gel purified.
  • the purified PCR product was sequenced using a standard protocol for ABI
  • 3700 sequencing 3700 sequencing. Briefly, triplicate reactions in forward and reverse orientation (6 total reactions) were prepared, each reaction containing 5 ⁇ l of gel purified PCR product as template.
  • 5' sequencing primer 5 ' CCC AGT CAC GAC GTT GTA AAA CG 3 ', SEQ ID NO: 54
  • the 3' sequencing primer was the sequence 5' GGT GGC GGC CGC AGG AAT TTT TTT TTT TTT TT 3', (SEQ ID NO: 56).
  • PCR was performed using the following thermal cycling program: 96°C, 2 minutes and 29 cycles of 96°C, 15 seconds; 50°C, 15 seconds; 60°C, 4 minutes.
  • Extended TOGATM assay was performed for each DST as described below.
  • PCR primers ("Extended TOGATM primers") were designed from sequence determined using one of three methods: (1) in suitable cases, the PCR product was isolated, cloned into a TOPO vector (Invitrogen) and sequenced on both strands; (2) in other cases, the TOGATM PCR product was sequenced using a modification of a direct sequencing methodology (Innis et al, Proc. Nat'l. Acad. Sci., 85: 9436-9440 (1988)) or (3) in many cases, the sequences listed for the TOGATM PCR products were derived from candidate matches to sequences present in available GenBank, EST, or proprietary databases.
  • PCR was performed using the Extended TOGATM primers and the Nl PCR reaction products as a substrate. Oligonucleotides were synthesized with the sequence G-A-T-C-G-A-A-T-C extended at the 3' end with a partial Mspl site (C-G-G), and an additional 18 adjacent nucleotides from the determined sequence of the DST.
  • the 5' PCR primer was G-A-T-C-G-A-A-T-C-C-G-G-C-T-A-A-T-A-T- T-G-A-T-A-T-C-T-T (SEQ ID NO:72).
  • This 5' PCR primer was paired with the fluorescence labeled universal 3' PCR primer (SEQ ID NO:23) in a PCR reaction using the PCR Nl reaction product as substrate.
  • the length of the PCR product generated with the Extended TOGATM primer was compared to the length of the original PCR product that was produced in the TOGA reaction.
  • SEQ ID NO:37 results for SEQ ID NO:37, for example, are shown in Figure 2.
  • the length of the PCR product corresponding to SEQ ID NO:37 (CLZ_43) was cloned and a 5 ' PCR primer was built from the cloned DST (SEQ ID NO:72).
  • the product obtained from PCR with this primer (SEQ ID NO:72) and the universal 3' PCR primer (SEQ ID NO:23) (as shown in the top panel) was compared to the length of the original PCR product that was produced in the TOGA reaction with mRNA extracted from the striatum/nucleus accumbens of mice treated with 7.5 mg/kg of clozapine for 12 days using a 5' PCR primer with parsing bases CTAA (SEQ ID NO:66) and the universal 3 ' PCR primer (SEQ ID NO:23) (as shown in the middle panel).
  • CTAA SEQ ID NO:66
  • SEQ ID NO:23 universal 3 ' PCR primer
  • a preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a sequence database, can be determined using the BLAST computer program based on the algorithm of Altschul and colleagues (Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990), "Basic local alignment search tool.” J. Mol. Biol. 215:403-410; Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D.J.
  • sequence includes nucleotide and amino acid sequences.
  • the query sequence can be either protein or nucleic acid or any combination therein.
  • BLAST is a statistically driven search method that finds regions of similarity between a query and database sequences. These are called segment pairs, and consist of gapless alignments of any part of two sequences. Within these aligned regions, the sum of the scoring matrix values of their constituent symbol pairs is higher than a level expected to occur by chance alone.
  • the scores obtained in a BLAST search can be inte ⁇ reted by the experienced investigator to determine real relationships versus random similarities.
  • the BLAST program supports four different search mechanisms:
  • Quantitation of the amount of target in the sample is accomplished by measuring the cycle number at which a significant amount of product is produced. The entire process is performed by the integrated software of the 7700 system. Primers for the Real Time PCR validation are selected by the integrated software package (Primer Express) accompanying the ABI PRISM 7700. Standards for normalizing the quantitation of gene levels were chosen from a panel of 5 mouse "housekeeping" genes. The normalization standard chosen was cyclophilin and was based on the similarity of expression across all sample templates.
  • FIG. 3 A-D compares the results from Real Time PCR validation (A) (as described below) to the TOGA TM result from three different experiments: the original clozapine experiment (B), a repeated clozapine experiment performed in duplicate (C), and a haloperidol experiment in duplicate (D).
  • TOGATM and Real Time PCR show that the DST CLZ_43 (SEQ ID NO:37) increases in expression in clozapine treated mice, while is not responsive to haloperidol treatment.
  • Table 5 lists the 5' and 3' primers used in these Real-Time PCR studies. Similar experiments were performed for DST CLZ_40 (SEQ ID NO: 12) and CLZ_5 (SEQ ID NO:2).
  • mice Male C57B1/6J mice (20-28 g) were housed as previously described in Example 1.
  • the same experimental paradigm used in Example 1 for clozapine treatment was used for the various analyses described below. Briefly, in the clozapine studies, the control group mice received a single injection of sterile saline (0.1 ml volume), or no injection, and were sacrificed after 45 minutes. The mice subjected to acute clozapine treatment were given a single intraperitoneal injection of clozapine (7.5 mg/kg) and sacrificed after 45 minutes or 7 hours, as described in Example 1. The mice subjected to chronic clozapine treatment received daily subcutaneous injections of clozapine (7.5 mg/kg) for 5 days, 12 days or 14 days.
  • FIG. 3 A-D compares the results from Real Time PCR validation (A) to the TOGA result from three different experiments: the original clozapine experiment (B), a repeated clozapine experiment performed in duplicate (C), and a haloperidol experiment in duplicate (D).
  • TOGA TM and Real Time PCR show that the DST CLZ_43 (SEQ ID NO:37) increases in expression in mice treated with clozapine for 12 days, while is not significantly responsive to haloperidol treatment for 14 days, relative to untreated mice.
  • Table 5 lists the 5' and 3' rimers used in these Real-Time PCR studies.
  • Real Time PCR confirmed that TOGA TM predicted a unique pattern by these two neuroleptics.
  • High stringency washes were carried out at 55°C for 2 hours in 0.5 X SSC/50% formamide/0.01 M ⁇ -mercaptoethanol, and then at 68°C for 1 hour in 0.1 X SSC/0.01 M ⁇ -mercaptoethanol/0.5% sarkosyl.
  • Slices were mounted onto gelatin- coated slides and dehydrated with ethanol and chloroform before autoradiography. Slides were exposed for 1-4 days to Kodak X-AR film and then dipped in Ilford K-5 emulsion. After 4 weeks, slides were developed with Kodak D19 developer, fixed, and counterstained with Richardson's blue stain.
  • Figure 4A-F demonstrates the pattern of CLZ_43 mRNA expression in coronal sections where A, B and C were sectioned at the level of the striatum (containing nucleus accumbens, Nacc, caudateputamen, Cpu, and neocortex, NC) and D, E, and F were sectioned at the level of the thalamus (Thai), hippocampus (Hipp), and hypothalamus (Hyp). A low level of expression was observed in the striatum, and treatment with either haloperidol or clozapine resulted in increased expression in the neocortex and in the striatum in mouse brain (B and C).
  • PCR products were gel purified, cloned into the TOPO plasmid vector (petlOOD TOPO, Invitrogen) and sequenced on both strands. Nucleotide sequences were determined by standard techniques.
  • Figure 5A-D compares the results from Real Time PCR validation (A) (as described in Example 1) to the TOGA result from three different experiments: the original clozapine experiment (B), a repeated clozapine experiment performed in duplicate (C), and a haloperidol experiment in duplicate (D).
  • A Real Time PCR validation
  • B original clozapine experiment
  • C repeated clozapine experiment
  • D haloperidol experiment in duplicate
  • the Real Time PCR shows that the mouse sequence homolog to human KIAA1451 (SEQ ID NO:79) increases in expression in mice chronically treated with clozapine (2.09-fold) or haloperidol (2.57-fold).
  • mouse KIAA 1451 -related sequence represents a neuroleptic responsive gene that is related, but distinct from the DST CLZ_43. This sequence also contains similarity to the same oxysterol binding protein family member as the DST.
  • an oligonucleotide designed from the human KIAA1451 sequence was used to isolate the remaining 5 'end of the human gene from an adult human brain cDNA plasmid library.
  • the target pool was a cDNA plasmid library created from adult human brain RNA.
  • the oligonucleotide sequence used for hybridization was 5' - AAC AAG TCC GTC CTG GCA TGG-3' (SEQ ID NO:51). The clone was isolated using the methods prescribed by the manufacturer of the GeneTrapper kit (Life Technologies, Inc.).
  • the capture oligonucleotides was end-labelled with biotin- 14-dCTP using terminal deoxynucloetidyl transferase and the cDNA plasmid pool was converted from double- stranded cDNA to single-stranded cDNA through the specific action of Genell protein and exonuclease III.
  • the single-stranded cDNA pool was combined with the end- labelled oligonucleotides, hybridized, mixed with strepavidin-coated magnetic beads, and plasmid/oligonucleotide hybrids were purified by magnetic separation.
  • the single-stranded plasmid DNA was released from the oligonucleotide and repaired back into a double-stranded plasmid using a fresh sample of the capture oligonucleotide and DNA polymerase. Plasmid DNA was prepared from bacteria transformed with the repaired plasmids and subjected to sequence analysis. Using this methodology, a 1717 b.p. cDNA clone (SEQ ID NO:68) was isolated that overlaps with the known human KIAA1451 sequence. This clone provides an additional (novel) 512 b.p. at the 5'end of the GenBank entry.
  • mice male C57B1/6J mice (20-28 g) were housed as previously described in Example 1 and divided into the following groups: 1) a control group, in which the mice were subcutaneusly implanted with one placebo pellet upon halothane anaesthesia; 2) an acute mo ⁇ hine group, in which the mice received a mo ⁇ hine intraperitoneal injection of 10 mg kg; 3) a chronic or tolerant group, in which mice were rendered drug-tolerant and dependent by means of subcutaneous implantation of a single pellet containing 75 mg of mo ⁇ hine free base for 3 days; and 4) a withdrawal group, in which the mice rendered tolerant to mo ⁇ hine were injected intraperitoneally with naltrexone 1 mg/kg.
  • mice Animals were sacrificed in their cages with CO 2 at 72 hours after placebo or mo ⁇ hine pellet implantation, or 4 hours after single injection of mo ⁇ hine, or 4 hours after administration of naltrexone to mo ⁇ hine-tolerant mice. Their brains were rapidly removed. The striatum, including the nucleus accumbens, and block of tissues containing the amygdala complex were dissected under microscope and collected in ice-cold RNA extraction buffer.
  • Figs. 6 and 7 show PCR products produced from mRNA isolated from the striatum/nucleus accumbens of mice treated with clozapine (Fig. 6) or mo ⁇ hine (Fig. 7).
  • the vertical index line indicates a PCR product of about 266 b.p.
  • the vertical index line indicates a PCR product of about 266 b.p. that is present in control cells, and whose expression differentially regulated in control striatum (PS), acutely treated striatum (AS), withdrawal striatum (WS), control amygdala (PA), acutely treated amygdala (AA), chronically treated amygdala (TA), and withdrawal amygdala (WA).
  • the expression of CLZ_40 product is greater in striatum than in amygdala.
  • CLZ_40 displays chronic-specific or withdrawal- specific regulation in both of these brain regions.
  • CLZ_40 is downregulated in withdrawal striatum but not acutely treated striatum.
  • CLZ_40 is slightly upregulated in acutely treated amygdala and increasingly upregulated in chronically treated amygdala and withdrawal amygdala.
  • Figure 9 is a graphical representation comparing the results of the clozapine treatment TOGATM analysis of clone CLZ_40 shown in Fig. 6 and the clozapine treatment Northern Blot analysis of clone CLZ_40 shown in Figure 8.
  • the Northern Blot was imaged using a phosphorimager to determine the amount of CLZ_40 mRNA in each clozapine-treated sample relative to the amount of mRNA in the control sample.
  • the clozapine treatment TOGA TM analysis shows correlation with the clozapine treatment Northern Blot analysis.
  • the single transcript of approximately 9 Kb was decreased in abundance after 45 minutes and 7 hr of clozapine treatment, consistent with TOGA.
  • CLZ_40 was further validated by Real Time PCR using cDNA from mice chronically (2 weeks) treated with clozapine or haloperidol (Figure 10A-D).
  • Figure 1 OA-D compares the Real Time PCR data (A) to the TOGA TM result from three different experiments: the original clozapine experiment (B), a repeated clozapine experiment performed in duplicate (C), and a haloperidol experiment in duplicate (D).
  • Real Time PCR analysis demonstrated a decrease of 0.37-fold in expression in clozapine treated mice and 0.66-fold decrease in haloperidol treated mice.
  • Figure 11 A-B shows in situ hybridization analysis, demonstrating CLZ_40 mRNA expression in the mouse brain. In situ hybridization was performed on free- floating sections as described in Example 2. Interestingly, CLZ_40 mRNA is specifically expressed in the nucleus accumbens and pyriform cortex (Fig. 11 A), and dentate gyrus (Fig. 1 IB), but is not detected in any other brain regions.
  • CLZ_40 (SEQ ID NO: 12) is of unknown identity.
  • the CLZ_40 DST has been PCR amplified and a larger cDNA clone that is approximately 1 Kb in length was obtained (SEQ ID NO: 13).
  • This sequence does match an EST in the Genbank database (AI509550) as shown in Table 4B.
  • a modified solid phase format of RACE (rapid amplification of cDNA ends) from mouse striatum cDNA was also utilized to obtain a 652 b.p. sequence (SEQ ID NO:80).
  • the observation that CLZ_40 is down-regulated with clozapine treatment suggests a potential association with the therapeutic effects of clozapine.
  • its highly unique gene expression pattern is like no other gene identified to date, and its presence in the nucleus accumbens may implicate CLZ_40 in a number of functional roles associated with this structure, namely limbic/mental behavior and addiction.
  • Addiction to opiates and other drugs of abuse is a chronic disease of the brain, most likely resulting from molecular and cellular adaptations of specific neurons to repeated exposure to opiates (Leshner, A., Science, 278, 45-47 (1997)).
  • An important neural substrate implicated in the opioid reinforcement and addiction is the mesolimbic system, notably the nucleus accumbens (Everitt, et al, Ann. N. Y. Acad. Set, 877, 412-438 (1999)). All highly addictive drugs, such as opiates, cocaine and amphetamines, produce adaptations in the neural circuitry of the nucleus accumbens, but the precise relationships are unknown.
  • CLZ_40 is a likely candidate for involvement in such mechanisms due to its specific expression in the nucleus accumbens. Elucidation of the biology underlying psychoses and addiction is key to understanding the underlying causes of such disorders and may lead to the development of more effective treatments, including anti-addiction medications.
  • Fig. 12 shows PCR products produced from mRNA isolated from the su ⁇ atum/nucleus accumbens of mice treated with clozapine for various lengths of time as described in Example 1.
  • the vertical index line indicates a PCR product of about 201 b.p. that is present in control cells, and whose expression increases when the striatum/nucleus accumbens of mice are treated with clozapine for 45 minutes, 7 hours, 5 days, 12 days, and 14 days.
  • the CLZ_5 clone (CACC 201; SEQ ID NO:2) corresponds with GenBank sequence X82648, which is identified as a mouse apolipoprotein D (apoD) sequence.
  • GenBank sequence X82648 is identified as a mouse apolipoprotein D (apoD) sequence.
  • Other corresponding apoD GenBank sequences include L39123 (mouse), X55572 (rat), NM_001647 (human).
  • Northern Blot analyses were performed to determine the effect of clozapine on apoD expression in mouse striatum/nucleus accumbens. Shown in Fig. 13, Northern Blot analysis was performed using 2 ⁇ g poly A enriched mRNA extracted from the striatum/nucleus accumbens of control mice and clozapine-treated mice, as described in Example 3.
  • a 160 bp insert of CLZ_5 25-100 ng was labeled with [ ⁇ - 32 P]-d CTP by oligonucleotide labeling to specific activities of approximately 5x10 8 cpm/ ⁇ g, added to the prehybridization solution and incubated for 1 hour.
  • apoD may be co-regulated by clozapine, in parallel with the mechanism of the clozapine therapeutic effects, and can serve as an indicator of clozapine bioactive levels.
  • Figure 14 is a graphical representation comparing the results of the clozapine treatment TOGA TM analysis of clone CLZ_5 (CACC 201) shown in Fig. 12 and the clozapine treatment Northern Blot analysis of clone CLZ_5 shown in Figure 13. The Northern Blot was imaged using a phosphorimager to determine the amount of apoD mRNA in each clozapine-treated sample relative to the amount of mRNA in the control sample. As can be seen, the clozapine treatment TOGA analysis shows correlation with the clozapine treatment Northern Blot analysis.
  • CLZ_5 is a strong candidate for an RNA encoding protein whose activity is involved in the benefit derived by patients from these two classes of neuroleptic drugs.
  • Figure 16A-C shows an in situ hybridization analysis, demonstrating the apoD expression in mouse brain.
  • the in situ hybridization was performed on free- floating sections (25 ⁇ M thick) as described in Example 2.
  • Fig. 16A shows CLZ_5 (apoD) mRNA expression in mouse anterior brain
  • 16B shows apoD mRNA expression in midbrain
  • 16C shows apoD expression in posterior brain. In all brain sections apoD is expressed by astroglial cells, pial cells, perivascular fibroblasts and scattered neurons.
  • FIG. 17A-I presents an in situ hybridization analysis, showing clone CLZ_5 (apoD) mRNA expression in mouse anterior (17A-C), mid (17D-F), and posterior (17G-I) brain following saline treatment (top row) or clozapine treatment (7.5 mg/kg) for 5 days (middle row) and 14 days (bottom row), using previously described methods.
  • Figure 18 A-H shows a darkfield photomicrograph demonstrating upregulated apoD mRNA expression in various brain regions, including the co ⁇ us callosum (cc, Fig. 18 A, E); caudate putamen (CPu, Fig. 18B, 18F); anterior commissure (aca, Fig. 18C, 18G); and globus pallidus (GP, Fig. 18D, 18H).
  • In situ hybridizations were perfomed as described above, using an antisense 35 S-labeled apoD riboprobe on brains from control (Fig. 18A-D) and clozapine-treated (Fig. 18E-H) animals.
  • the observed upregulation of apoD was due to an increase in the amount of apoD expressed per cell.
  • Figure 19A, B shows a darkfield photomicrograph demonstrating upregulated apoD mRNA expression in the internal capsule (ic).
  • Figure 19C, D shows a brightfield view of the optic tract (opt) demonstrating upregulation of apoD expression in oligodendrocytes.
  • In situ hybridizations were perfomed as described above, using an antisense 35 S-labeled apoD riboprobe on brains from control (19A, C) and clozapine-treated (19B, D) animals.
  • the cells prominantly expressing apoD in the optic tract have a box-like mo ⁇ hology and are lined up in a serial array, presumably along axonal tracts.
  • White matter tracts comprise nerve fiber bundles connecting different regions of the brain.
  • the predominant cells in these regions are asfrocytes and oligodendrocytes, both of which have been shown to express apoD (Boyles et al, J Lipid Res 31:2243-2256 (1990); Navarro et al., Neurosci Lett 254:17-20 (1995); Provost et al., J Lipid Res 32 (1991)).
  • apoD riboprobe 35 S- labeled apoD riboprobe in combination with either an antibody specific for an astrocyte marker, glial fibrillary acidic protein (GFAP), or an antibody specific for an oligodendrocyte marker, 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNP) (Boehringer Mannheim, Germany).
  • GFAP glial fibrillary acidic protein
  • CNP 2', 3'-cyclic nucleotide 3'-phosphodiesterase
  • the immunoreaction was detected with Nectastain ABC TM kit (Vector Laboratory, Inc., Burlingame, CA) according to the manufacturer's instructions.
  • Free floating brain sections were incubated with blocking solution (4% bovine serum albumin in 0.1% Triton X-100/PBS) for 2 hours at room temperature, followed by incubation with anti-GFAP or anti-C ⁇ P antiserum (dilution 1:500) in blocking solution for 16-20 hours at 4°C. Sections were then washed with 0.1% Triton X-100/PBS and incubated with secondary biotinylated antibody (1:200 dilution in blocking solution) for 2 hours at room temperature. The sections were then washed with 0.1% Triton X- 100/PBS, incubated for 1 hour with ABC reagent (1 :1 in blocking solution) and finally washed with 0.1% Triton X- 100/PBS. Enzymatic development was performed in 0.05% diaminobenzene in PBS containing 0.003%> hydrogen peroxide for 3-5 minutes.
  • blocking solution 4% bovine serum albumin in 0.1% Triton X-100/PBS
  • Fig. 20 shows sections of striatum and optic tract in control and clozapine- treated animals. Thick arrows indicate the co-localization of GFAP and apoD, while thin arrows indicate the expression of apoD alone.
  • Fig. 20A, B shows that in untreated striatum, many GFAP-positive cells in both gray and white matter regions are positive for apoD.
  • Fig. 20D, E shows that in brain from clozapine-treated animals, an increase in the amount of apoD was observed in a small subset of GFAP- positive cells in the striatum.
  • Fig. 20C F shows GFAP and apoD co-localization in the optic tract in control (20C) and clozapine-treated (20F) animals. While some asfrocytes express apoD rnR ⁇ A, the cells responsible for the predominant apoD transcript upregulation did not label with GFAP and thus are likely oligodendrocytes.
  • Fig. 20G, H shows apoD immunohistochemistry with an anti-human apoD primary antibody (Novocastra, Newcastle, UK) in the optic tract of control saline (20G) and clozapine-treated animals (20H).
  • FIG. 21 shows a Northern Blot analysis of clone CLZ_5 expression in cultured glial cells treated with clozapine (100 nM and 1 ⁇ M) for 1 day or 7 days. Glial cell cultures were produced from postnatal (day 2) rats.
  • 20 ⁇ g of total cytoplasmic RNA from glial cell cultures were electrrophoresed on a 1.5% agarose gel containing formaldehyde, blotted, and probed as previously described.
  • apoD mRNA levels were down-regulated in mixed glial cell cultures treated with clozapine (both 100 nM and 1 ⁇ M) for 1 week, suggesting that perhaps neurons and glia display different mechanisms for apoD regulation.
  • TOGATM methodology, Northern blot analyses, Real Time PCR, and in situ hybridization studies have demonstrated an increase in apoD mRNA expression in both white and gray matter regions of mouse brain in response to chronic clozapine administration.
  • Co-localization studies, combining in situ hybridization and imunohistochemistry methods have revealed that apoD mRNA levels are increased in both neurons and glial cells with clozapine administration. The evidence indicates that the glial cells responsible for the most dramatic increases in apoD expression are primarily oligodendrocytes, but a subset of asfrocytes also have increased apoD expression after clozapine treatment.
  • Real-Time PCR analysis suggested that apoD expression was also affected by haloperidol treatment.
  • a novel oxysterol binding protein (CLZ_43, Example 2) and apoD are among the few modulated by neuroleptic drugs strengthens the hypothesis that schizophrenia is a disease of brain sterol homeostasis, and thus may have etiologies as diverse as atherosclerosis.
  • the brain has by far more cholesterol and 24S-hydroxysterol than any organ other than the adrenal glands, and the special importance of the membrane activities of neurons and their myelinating cells are self-evident.
  • the lipid bilayer of the membrane is made up of glycerolphopholipids and cholesterol, and variations in composition and hydrocarbon chain saturation state determine membrane order and fluidity.
  • Example 6 describes studies investigating a potential role for apoD in the neuropathology of Alzheimer's disease.
  • apoD mRNA expression was measured in transgenic mice expressing mutated human amyloid precursor protein under control of platelet-derived growth factor promoter (PDAPP mice), and the findings suggest that, although increases in apoD expression are a normal feature of brain aging, super-increases may represent a glial cell compensatory response to beta-amyloid deposition in Alzheimer's disease.
  • PDAPP mice platelet-derived growth factor promoter
  • ApoD was initially identified as a constituent of plasma high-density lipoproteins (HDLs), which also contain phospholipids, cholesterol and fatty acids (McConathy et al., Fed. Eur. Biochem. Soc. Lett, 37: 178 (1973)).
  • HDLs plasma high-density lipoproteins
  • apoD is thought to play a role in reverse cholesterol transport, the removal of excess cholesterol from tissues to the liver for catabolism (Oram et al., J. Lipid. Res., 37: (1996)).
  • apoD is major protein component in cyst fluid from women with human breast cystic disease (Balbin et al., Biochem.
  • ApoD has also been shown to bind arachidonic acid (Morais-Cabral et al, FEBS Lett, 366: 53 (1995)) implicating it in functions associated with cell membrane remodeling and prostaglandin synthesis.
  • arachidonic acid Morais-Cabral et al, FEBS Lett, 366: 53 (1995)
  • apoD concentrations are increased 500-fold (Boyles et al., J. Biol Chem., 265: 17805 (1990)).
  • apoD may play an important role in psychotic disease. It is widely believed that imbalances in basal ganglia circuitry contribute to psychotic behaviors and that blockade of specific receptors in these regions is responsible for neuroleptic action. The neuronal increases in apoD mRNA expression observed in neurons of the striatum and globus pallidus are consistent with this hypothesis.
  • the internal capsule consists of massive nerve fibers connecting the thalamus to the cortex and is an area of convergence for the fiber tracts running transversely through the striatum.
  • the thalamus is a relay station for virtually all information passing to the cortex and coordinated cortico-thalamic activity is essential for normal consciousness. Recent theories have associated psychotic behavior with disruptions in cortico-thalamic oscillations.
  • An upregulation of apoD expression in the internal capsule may play a role in restoring the proper balance of neuronal communication.
  • ApoD is a constituent of plasma high-density lipoproteins (HDLs), which also contain phospholipids, cholesterol and fatty acids. While not much is known about HDL compared to the other plasma lipoproteins, LDL and VLDL, it is widely believed that HDLs protect against cardiovascular disease by removing excess cholesterol from cells of arterial walls. This removal involves the direct interaction of HDL lipoproteins with plasma membrane domains and subsequent transport to the liver for catabolism (Oram, et al., J. Lipid Res., 37, 2473-2491 (1996)).
  • apoD is synthesized and secreted by cultured asfrocytes, which secretion has been shown to increase in the presence of cholesterol derivatives (Patel, et al, Neuroreport 6, 653- 657 (1995)). Further, it has also been demonstrated that apoD levels are increased in Niemann Pick Disease, type C, which is associated with elevated levels of cholesterol. These studies provide evidence of a functionally significant role for apoD in cholesterol transport in the CNS. In addition to the studies correlating cholesterol levels and psychotic behavior, other studies have found a correlation between cholesterol levels and treatment with neuroleptics.
  • Membrane phospholipids act as precursors in numerous signaling systems (e.g., inositol phosphates, arachidonic acid, platelet activation factors, and eicosaniods) and comprise the membrane environment for neurotransmitter-mediated signal transduction.
  • signaling systems e.g., inositol phosphates, arachidonic acid, platelet activation factors, and eicosaniods
  • Alterations in plasma membrane structure and function may result from the altered content and distribution of membrane lipids and fatty acids, such as arachidonic acid.
  • Arachidonic acid is released by the action of numerous phosphohpase enzymes, primarily phosphohpase A2, and is a substrate for prostglandins and leukotriene synthesis. While the molecular mechanisms underlying abnormalities in the complex system of phospolipid biochemistry are not known, several groups have demonstrated an increase in phosphohpase A2 activity in the plasma and brains of schizophrenic patients (Gattaz et al, Biol. Psychiatry., 22, 421- 426 (1987); Ross et al., Arch. Gen.
  • Psychiatry., 54, 487-494 (1997); Ross et al, Brain Research, 821, 407-413 (1999)).
  • plasma phosphohpase A2 levels have been shown to be decreased after neuroleptic therapy (Gattaz et al., Biol. Psychiatry, 22, 421-426 (1987)).
  • Other molecular candidates implicated in psychotic disease include phosphohpase C enzymes, diacyl glycerol kinases, and inositol phosphates (Horrobin et al., Prostaglandins, Leukotrienes and Essential Fatty Acids, 60, 141-167 (1999)).
  • apoD has been shown to specifically bind arachidonic acid.
  • ApoD is an atypical apolipoprotein in that it does not share sequence homology with other apolipoproteins (Weech et al., Prog. Lipid Res., 30, 259-266 (1991)) but, rather, is a member of the lipocalin superfamily of proteins, which function in the transport of small hydrophobic molecules, including sterols, steroid hormones, and arachidonic acid (Balbin et al., Biochem. J., 271, 803- 807 (1990); Dilley et al., Breast Cancer Res.
  • apoD can affect fatty acid composition, cholesterol levels and membrane phospholipids, all of which will affect plasma membrane composition and structure. Also, since apoD specifically binds cholesterol, arachidonic acid and other lipids, alterations in the levels of apoD can affect lipid metabolism and signal transduction by affecting substrate availability for these pathways.
  • apoD may have a chromosomal linkage with schizophrenia.
  • the chromosomal location of apoD is 3q26.
  • Genetic studies have implicated a potential association between schizophrenia and chromosome 3q, however the linkage is relatively inconsistent (reviewed by Maier, et al, Curr. Opin. Psych., 11, 19-25 (1998)).
  • a serotonin sub-type such as 5HT 2a and 5HT 2c may provide a pharmacological mechanism for clozapine's effect on apoD expression.
  • Preliminary results demonstrate that treatment with ketanserin and mesulergine, 5HT a/2c and 5HT 2c -selective antagonists respectively, results in an apparent upregulation of apoD mRNA expression in mouse brain.
  • the striatum expresses a number of 5HT receptor subtypes, including the 5HT 2c , which subtype may mediate clozapine's effect on apoD expression.
  • cultured glial cells or asfrocytes do not appear to express 5HT 2c receptors.
  • the downregulation observed in these cells may reflect actions at a different 5HT subtype, such as 5HT 2a , or a different receptor.
  • 5HT 2a a different receptor
  • ketanserin has been associated with a decrease in total cholesterol levels and an upregulation of another apolipoprotein, apo Al (Loschiavo, et al., Int. J. Clin. Pharmacol. Ther. Toxicol, 28, 455-457 (1990)).
  • apo Al apolipoprotein
  • the similar effects observed by both ketanserin and clozapine suggest that they may be working through the same receptor subtype(s).
  • apoD mRNA levels are increased by clozapine links apolipoproteins and the mechanism of action of neuroleptic drugs.
  • the proposed role of apoD in CNS lipid transport combined with the recent evidence that schizophrenia and other neuropsychiatric illnesses are accompanied by abnormalities in lipid metabolism, suggest that apoD could play an important role in the action of clozapine.
  • apoD is regulated by chronic antipsychotic drug administration and the pattern of apoD expression in the brain suggests that apoD may play an important role in psychotic disease.
  • This example demonstrates that apoD expression is increased in the dorsolateral prefrontal cortex region (BA9) and caudate of the brains of schizophrenic and bipolar human subjects compared with control human subjects.
  • BA9 dorsolateral prefrontal cortex region
  • the combined results suggest that apoD is a marker for neuropathology associated with psychiatric disorders and therefore can be used to target abnormalities in specific anatomical brain regions.
  • Membrane homogenates were prepared from various brain regions (prefrontal cortex, occipital cortex, substantia nigra, cerebellum, hippocampal formation, and caudate) of control, schizophrenic or bipolar subjects by homogenization in Tris buffer (20 mM Tris-HCl, 0.2 mM EGTA, 0.1 mM EDTA, pH 7.4) including 3x "complete" protein inhibitor tablets (Boehringer Mannheim). Aliquots of the membrane homgenates (50 ⁇ g total protein/lane) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 12% acrylamide gel.
  • Tris buffer (20 mM Tris-HCl, 0.2 mM EGTA, 0.1 mM EDTA, pH 7.4
  • 3x "complete" protein inhibitor tablets Boehringer Mannheim.
  • the gels were transferred to nitrocellulose membranes, blocked with 5%> milk in T- TBS (Tris-buffered saline/0.1% Tween-20, pH 7.5) and then probed with a monoclonal antibody directed against human apoD) (1:500 dilution) (Novacastra).
  • Enhanced chemiluminescence ECL, Amersham, Arlington Heights, IL was used to detect immunoreactivity and blots were visualized by exposure to autoradiography film.
  • Figure 22 shows Western blot analyses of various brain sections in control human subjects.
  • Western blots containing 50 ⁇ g total protein per lane were probed with a monoclonal antibody directed against apoD.
  • Enhanced chemiluminescence (ECL) was used to detect immunoreactivity and blots were visualized by exposure to autoradiography film.
  • Western blot analysis using an antibody specific to apoD revealed widespread distribution of apoD protein in various human brain regions, including prefrontal cortex (BA9 and BA10), components of the hippocampal formation (CA1, CA3, dentate gyrus, subiculum, parahippocampal gyrus) and basal ganglia (caudate and substantia nigra) (see Fig. 22).
  • ApoD immunoreactivity was detected in all brain regions tested, with the highest level of expression observed in the substantia nigra (SN). A major band of 29 kDa was observed in all regions examined, however, an additional band of approximately 22 kDa was observed in other brain regions, primarily Brodman's Area 10, substantia nigra, CA1 and subiculum.
  • FIG. 23 A-B are Western blot analyses showing apoD expression levels in the dorsolateral prefrontal cortex, Brodman's Area 9 (BA9), of eight schizophrenic subjects (Sch-lto Sch-8) and eight age- and sex-matched control subjects (Con-lto Con-8).
  • Brodman's Area 9 is a region previously implicated in the pathophysiology of schizophrenia (for review, see Goldman-Rakic et al., Schiz. Bull, 23: 437 (1997)).
  • Western blots containing 50 ⁇ g total protein per lane were probed with a monoclonal antibody directed against apoD.
  • Enhanced chemiluminescence (ECL) was used to detect immunoreactivity and blots were visualized by exposure to autoradiography film.
  • ECL Enhanced chemiluminescence
  • Two monoclonal antibodies to apoD from Signet Laboratories, Inc. (Dedham, MA, USA) were used in a sandwich assay.
  • .Microtitre high capacity binding plates Costar
  • the wells were washed 4x with T-TBS, blocked with 5% bovine serum albumin in T-TBS for 1 hour at room temperature and then washed again 4x with T-TBS.
  • An aliquot (50 ⁇ l) of the various tissue homogenates (50 ⁇ g total protein) was added and incubated for 1 hr at room temperature.
  • the wells were washed 4x with T-TBS, and then 50 ⁇ l of a second, HRP-conjugated, apoD antibody was added to each well and incubated 1 hour at room temperature. After extensive washing with T-TBS, 50 ⁇ l of TMB substrate system (Sigma Chemical Co.) was added to allow color formation.
  • TMB substrate system Sigma Chemical Co.
  • Figure 24C shows ELISA assay measurements performed in the caudate from the same schizophrenic patients and control subjects used to determine apoD levels in the BA9 regions.
  • apoD apoD expression is significantly increased (1.9- to 3.9- fold) in dorsolateral prefrontal cortex of schizophrenic and bipolar patients, which is a brain region previously implicated in the pathophysiology of schizophrenia.
  • apoD expression is not increased in the occipital cortex, a region with no association to schizophrenia. Numerous experimental and clinical studies have provided evidence of pathophysiological changes in the prefrontal cortex of patients with schizophrenia.
  • apoD expression in the occipital cortex (BA18), substantia nigra, cerebellum or hippocampus, indicating regional specificity for apoD expression induction.
  • components of bipolar disorder have also been associated with abnormal functioning of the prefrontal cortex (Blumberg et al, Am JPyschiatiy, 156: 1986-1988 (1999); Knable, M.B., Schizophr Res, 39: 149-152 (1999); Drevets et al., Mol Psychiatry, 3: 220-226 (1998)).
  • the increases observed in the caudate are also consistent with studies implicating basal ganglia structures in the pathophysiology of psychiatric disorders.
  • apoD expression is elevated under apparently diverse conditions, it is possible that apoD expression represents a non-specific response to stress or pathological insult.
  • apoD upregulation observed after CNS insult in the rodent studies and the regional specificity of apoD induction observed in human disease and mouse models (Niemann-Pick)
  • apoD is a region-specific marker for active pathological processes.
  • Our findings that clozapine induced apoD accumulation in rodent brains had suggested the simple hypothesis that increases apoD may be beneficial to patients with neuropsychiatric disorders.
  • the present findings suggest that apoD accumulation might be a natural response to regional neuropathology, and that one reason clozapine is an effective antipsychotic drug is via its ability to augment increases in apoD already present in the brain.
  • composition and hydrocarbon chain saturation state determine membrane order and fluidity, and these properties affect the binding and function of extrinsic membrane proteins and second messenger signaling.
  • changes in the levels of apoD can potentially affect membrane phospholipid composition, by increasing or decreasing transport and uptake of these membrane constituents.
  • Phospholipids play a critical role in almost every function of the cell membrane and its metabolic products are crucial for cellular functions and cell-to-cell communication.
  • Arachidonic acid acts as a second messenger in several neurotransmitter systems, including the action of basic fibroblast growth factors that are critical for normal brain development. Synaptic organization would also dependent upon the integrity of the membrane structure. Recent studies have demonstrated increases in various presynaptic proteins (Gabriel et al., Arch Gen Psychiatry, 54: 559-566 (1997)), and synapsin and synaptophysin, two synaptic vesicle-associated proteins (Browning et al., Biol Psychiatry, 34: 529-535 (1993); Honer et al, Neurosci, 91: 1247-1255 (1999), in cerebral cortex of schizophrenic subjects.
  • apoD levels in serum samples were measured in serum samples of schizophrenic subjects and from brain tissue obtained post-mortem from schizophrenic and bipolar subjects and subjects with no history of psychiatric illness (controls) using both Western blot and ELISA analyses. ApoD concentrations were determined in the serum from consenting neuroleptic-free patients, patients receiving typical neuroleptic drugs and patients enrolled in the clozapine monitoring system at the Mental Health Research Institute.
  • schizophrenic patients were deemed neuroleptic free if they had not received neuroleptic drugs orally for 1 month or by depot injection for 3 months before blood collection.
  • AA-enriched phospholipids have been observed in cultured fibroblasts in chronic and in first episode schizophrenic patients (Mahadik et al., Schizophrenia Res, 13: 239-247 (1994); Mahadik et al., Psychiatry Res, 63: 133-142 (1996)).
  • apoD levels are low in the serum of schizophrenic subjects, but elevated in the dorsolateral prefrontal cortex and caudate of schizophrenic and bipolar subjects.
  • apoD may be a compensatory region-specific marker for a neuropathological process that is initiated because of systemic lipid metabolism insufficiencies.
  • Example 4 The mouse studies described above (Example 4) showed that apoD is regulated by chronic antipsychotic drug administration, and Example 5 described an increase in apoD expression in the prefrontal cortex of schizophrenic and bipolar human subjects.
  • Example 5 described an increase in apoD expression in the prefrontal cortex of schizophrenic and bipolar human subjects.
  • apoD is a marker for neuropathology associated with psychiatric disorders and therefore can be used to target abnormalities in specific anatomical brain regions.
  • This example describes studies investigating a potential role for apoD in the neuropathology of Alzheimer's disease.
  • This example describes the measurement of ApoD mRNA expression in transgenic mice expressing mutated human amyloid precursor protein under control of platelet-derived growth factor promoter (PDAPP mice), and the findings suggest that, although increases in apoD expression are a normal feature of brain aging, super- increases may represent a glial cell compensatory response to beta-amyloid deposition in Alzheimer's disease.
  • PDAPP mice platelet-derived growth factor promoter
  • AD Alzheimer's disease
  • a ⁇ amyloid ⁇
  • APP amyloid ⁇ precursor protein
  • apolipoprotein E a 34 kDa very low-density protein that has been implicated in the pathogenisis of AD.
  • apoE apolipoprotein E
  • ⁇ 2, ⁇ 3 and ⁇ 4 three major allelic variations in the apoE gene ( ⁇ 2, ⁇ 3 and ⁇ 4) exist and these encode three protein isoforms. It is now well established that inheritance of the ⁇ 4 allele greatly increases the risk for developing late-onset familial and sporatic AD (Strittmatter et al., 1993).
  • apoE mRNA and protein levels have been shown to be elevated in brains of Alzheimer's subjects (Yamada et al., 1995, Yamagata et al., 2001), and apoE immunoreactivity has been localized not only to the senile plaques, but also to vascular amyloid and the neurofibrillary tangles of AD (Poirier, 2000). It has also been demonstrated that apoE is essential for beta-amyloid deposition in a mouse model of AD (Bales et al, 1999). In addition to apoE, other apolipoproteins have been implicated in AD suggesting perhaps a common pathway of lipid homeostasis in the pathology and progression of the disease.
  • amyloid plaques in AD patients have also been shown to be immunoreactive for apoA-1, apoJ and apoB (Harr et al, 1996, Namba et al, 1992, Calero et al, 2000).
  • increased levels of apoJ have been detected in the cortex and hippocampus of Alzheimer's subjects (Lidsfrom et al, 1998) and abnormal levels of apoB and apoA-1 have been reported in plasma from Alzheimer's subjects (Caramelli et al, 1999, Merched et al, 2000).
  • ApoD has also been associated with AD (Kalman et al, 2000, Belloir et al, 2001, Terrisse et al, 1998).
  • ApoD is a 29 kDa glycoprotein that, like apoE and apoJ, is synthesized in cells within the CNS (reviewed by Rassart et al, 2000).
  • apoD is composed primarily of antiparallel ⁇ -sheets, hence shares a similar structure to the lipocalin superfamily of lipid-binding proteins (Rassart et al, 2000).
  • apoD has been shown to bind hydrophobic molecules, such as steroid hormones, retinoids, heme-related compounds and arachidonic acid (Rassart et al, 2000).
  • hydrophobic molecules such as steroid hormones, retinoids, heme-related compounds and arachidonic acid
  • the function of apoD in the CNS is not clear, but it is thought to function in maintenance and repair after CNS insult or in response to CNS pathology.
  • Recent studies have demonstrated increased levels of apoD in the CSF of AD and other neurological disorders and increases in the hippocampus and cortex of AD subjects (Terrisse et al, 1998, Kalman et al, 2000, Belloir et al, 2001).
  • Examples 4 and 5 of the current embodiment demonstrated increased apoD expression in prefrontal cortex and caudate of schizophrenia and bipolar disorder subjects (Thomas et al, 2001b). These cumulative findings have led to the hypothesis that apoD is a marker for brain regions that undergo neuropathology as a component of various human neurological disorders.
  • mice overexpressing the human APP gene or APP mutations have been developed (Higgins et al, 1994, Mucke et al, 1994, Games et al, 1995)).
  • One model, the PDAPP mouse harbors a mutation in APP directing a valine to phenylalanine change at position 717 (APPV717F) resulting in an ove ⁇ roduction of the highly amyloidogenic A ⁇ (1-42) relative to other A ⁇ peptides.
  • This mouse exhibits many prominent age-dependent pathological and behavioral features of AD, including progressive neuropathology, amyloid beta deposition, neuritic plaques, astrocytosis and microgliosis and synaptic loss (Games et al, 1995, Chen et al, 1998, Chen et al, 2000, Masliah et al, 1996).
  • apoD mRNA expression in brains of young and aged PDAPP transgenic mice Transgenic mice expressing human mutant APP (APP V717F) have been described previously (Games et al, 1995, Chen et al, 1998).
  • mice were developed using a platelet-derived growth factor promoter driving a hBAPP minigene encoding the 717 V ⁇ F mutation associated with familial AD (Games et al, 1995).
  • Mice were fifth generation female heterozygous PDAPP line 109 mice produced on a Swiss Webster X B 6 D 2 Fi (C57BL/6 x DBA/2) outbred background (Games et al, 1995, Masliah et al, 1996).
  • aged (26 month) and young (6 month) groups of female PDAPP mice and non-Tg littermates were utilized (provided by Elan Pharmaceuticals).
  • mice exhibit many prominent age-dependent pathological features of AD, including progressive neuropathology, amyloid beta deposition, neuritic plaques, gliosis and decreased synaptic and dendritic densities (Chen et al, 2000, Chen et al, 1998, Masliah et al, 1996, Games et al, 1995).
  • mice were anesthetized with halothane and perfused with cold phosphate buffered saline (PBS; pH 7.5) followed by 4% paraformaldehyde in PBS. Brains were removed and post-fixed in 4% paraformaldehyde overnight, then cryoprotected in 30% sucrose in paraformaldehyde for 24 hr. In situ hybridization was performed on free-floating sections as described previously (Example 2).
  • PBS cold phosphate buffered saline
  • Figure 27 A represents tissue slices taken at the level of the caudatoputamen (CP), demonstrating gene expression in the co ⁇ us callosum (cc) and septal white matter tracts (sp).
  • Figure 27B represents tissue slices taken at the level of of the globus pallidus (GP) demonstrating gene expression in the hippocampal fimbria (fi) and co ⁇ us callosum (cc).
  • Figure 27C,D represent tissue slices taken at the level of the hippocampus (Hipp) and thalamus (Th) demonstrating gene expression in the co ⁇ us callosum (cc).
  • AD neurodegenerative changes in the PDAPP mice are observed in the hippocampus and cortex, two brain structures implicated in the pathophysiology of AD.
  • a ⁇ deposits and aggregates begin to form in the PDAPP mice at around 8 months and, by one year of age, A ⁇ deposits are common in the hippocampus and in the frontal and cingulate cortices (Johnson- Wood et al, 1997). These mice also develop behavioral deficits that are likened to the cognitive decline seen in AD (Chen et al, 2000).
  • AD is generally considered to affect grey matter regions, histological studies have demonstrated pathological changes in white matter (Bran and Englund, 1986, Rose et al, 2000).
  • Loss of white matter integrity can be responsible for loss of connectivity in AD and consequential decline in cognitive functions (Bran and Englund, 1986).
  • the co ⁇ us callosum is the largest white matter structure in the brain connecting the neocortex of each side and is responsible for normal interhemispheric communication.
  • the hippocampal fimbria is another white matter structure important for connectivity of the hippocampus. Afferents from the septum enter the hippocampal formation via the fimbria and are distributed to both the hippocampus and dentate gyrus. Accordingly, increases in apoD expression in the septal white matter tracts were also observed. Super-expression of apoD in the white matter regions may have several implications regarding the effect of amyloid plaques in the pathology of AD.
  • AD Alzheimer's disease
  • astrocytes and microglia are a common feature of other neurodegenerative disorders and CNS pathology. Gliotic changes are observed in PDAPP mice similar to those observed in AD (Games et al, 1995, Chen et al, 1998).
  • a ⁇ peptides, including A ⁇ l- 42, can induce profound glial cell activation, suggesting that the A ⁇ deposition in this model may be responsible for the observed gliotic effects.
  • the increased expression of apoD in astrocytes may indicate a response associated with A ⁇ deposition-induced astrocytosis.
  • apoD is elevated in response to microglial activation (Monica Carson, personal communication).
  • oligodendrocytes which are the cells responsible for myelin synthesis. ApoD expression in these cells may reflect a dysfunction in myelin or axonal integrity resulting from A ⁇ deposition and plaque formation. This is consistent with white matter deficits associated with AD. Histological studies have shown pathological changes, such as a loss of axons and oligodendrocytes together with a reactive astrocytosis in the white matter regions of AD subjects (Rose et al, 2000, Bran and Englund, 1986).
  • AD Alzheimer's disease
  • apoD may have a similar function in the CNS as the other apoproteins, it does not share a similar protein structure as the other family members and does not bind cholesterol with high affinity, hence may have a unique effect via binding of different ligands.
  • ApoD may be involved in the binding of steroids or fatty acids released upon CNS insult, or the transport of lipid molecules necessary for cellular regeneration, and therefore may function in CNS maintenance and tissue repair.
  • apoD is a marker for neuropathology associated with psychiatric disorders and therefore can be used to target abnormalities in specific anatomical brain regions.
  • Example 1 The same experimental paradigm used in Example 1 for clozapine treatment was used for the TOGA analyses.
  • This experiment describes the validation of two additional DSTs by Northern analysis (CLZ_38 and CLZ_44), and characterization of expression patterns by in situ hybridization on 10 DSTs (CLZ_3, CLZ_16, CLZ_17, CLZ_24, CLZ_26, CLZ_28, CLZ_34 , CLZ_ 38, CLZ_44 , CLZ_64) in mouse CNS.
  • In situ hybridization was performed on free-floating coronal sections (25 ⁇ M thick) with an 35 S-labeled, single-stranded antisense cRNA probe specific for each DST using the methods described in the above examples. The regional distributions for these are summarized in Table 9.
  • a subset of clones exhibit expression in specific brain regions, hence are of particular interest. These include CLZ_3, CLZ_17, CLZ_38, CLZ_40 (Example 3), CLZ_43 (Example 1), and CLZ_44.
  • CLZ_24, CLZ_26 and CLZ_28 displayed relatively enriched expression in the cortex. Since specific expression of genes in a certain brain region reflects an association with the functional specialization of the region, these studies are useful to determine the role of specific genes and their contribution to brain function.
  • the striatum (dorsal striatum) is responsible for motor and movement functions, while the nucleus accumbens (ventral striatum) and other limbic regions are involved in cognitive and emotional behavior, as well as reward and reinforcement.
  • the identification of genes that are specifically expressed in a particular brain region will elucidate the mechanisms of brain function.
  • Figure 30A-B is an in situ hybridization analysis using an antisense cRNA probe directed against the 3' end of CLZ_3, showing the pattern of CLZ_3 mRNA expression in a coronal section through the hemispheres at level of hippocampus (Fig 30 A) and cross section through midbrain (Fig. 30B) in mouse brain.
  • CLZ_3 mRNA is expressed in the cortex, thalamus, hippocampus, striatum, and amygdala.
  • CLZ_24 (SEQ ID NO: 7) is up-regulated by clozapine treatment.
  • Table 2 shows that CLZ_24 is an EST isolated from rat tissue.
  • in situ hybridization analysis using an antisense cRNA probe directed against the 3' end of CLZ_24 were performed to show the pattern of CLZ_24 mRNA expression in mouse anterior brain (Fig. 33B) and posterior brain (Fig. 33A).
  • Figure 33A-B shows an in situ hybridization analysis using an antisense cRNA probe directed against the 3 ' end of CLZ_24, showing the pattern of CLZ_24 mRNA expression in a coronal section through the hemispheres (Fig. 33 A) and cross section through the brainstem (Fig. 33B) in mouse brain.
  • CLZ_24 mRNA is ubiquitously expressed in the cortex.
  • Figure 34A-B is an in situ hybridization analysis using an antisense cRNA probe directed against the 3' end of CLZ_26, showing the pattern of CLZ_26 mRNA expression in a coronal section of the hemispheres at the level of hippocampal formation (Fig. 34A) and coronal section of the hemispheres at the level of striatum (Fig. 34B) in mouse brain. As shown, CLZ_26 mRNA is ubiquitously expressed in the cortex.
  • Figure XA-B is an in situ hybridization analysis using an antisense cRNA probe directed against the 3' end of CLZ_28, showing the pattern of CLZ_28 mRNA expression in a coronal section through the hemispheres at the level of hippocampus (Fig. 35 A) and coronal section through the posterior region of hemispheres (Fig. 35B) in mouse brain.
  • CLZ_28 mRNA is expressed ubiquitously in the cortex.
  • CLZ_34 (SEQ ID NO: 9) is upregulated with clozapine treatment at 45 minutes and 7 hours, but decreases to control level by day 5 and remains at about control level for as long as 12 days, showing a slight increase at day 14.
  • CLZ_34 corresponds with GenBank sequence UO8262, which is identified as a rat N-methyl-D-aspartate receptor/NMD AR1 -2a subunit (NMDAR1) (Table 2 and 3).
  • the NMDAR1 receptor is a glutamate receptor involved in the processes underlying learning and memory.
  • blockade of glutamate actions by noncompetitive (e.g. MK801 and dextrometho ⁇ han) and competitive (e.g.
  • NMDA receptor antagonists blocks or reduces the development of mo ⁇ hine tolerance following long term opiate administration (Trajillo et al, Science, 251, 85-87, (1991); Elliott et al, Pain, 56, 69-75 (1994); Wiesenfeld-Hallin, Z., Neuropsychopharm., 13, 347-56 (1995)).
  • CLZ_34 which has high homology with an NMDA receptor is interesting in view of the ability of NMDA antagonists to block the development of tolerance to opioids.
  • FIG. 36A-B is an in situ hybridization analysis using an antisense cRNA probe directed against the 3' end of CLZ_34, showing the pattern of CLZ_34 mRNA expression in a coronal section through the hemispheres at the level of hippocampus (Fig 36 A) and cross section through the midbrain (Fig. 36B) in mouse brain.
  • CLZ_34 mRNA is ubiquitously expressed.
  • CLZ 38 Table 2 shows that CLZ_38 (SEQ ID NO: 11) is an oligodendrocyte-specific protein mRNA.
  • Northern blot analyses were performed to determine the pattern of expression in the striatum/nucleus accumbens of control mice and mice treated with clozapine for 45 minutes, 7 hours, 5 days, and 2 weeks.
  • Figure 37 is a graphical representation of the described Northern blot analyses. As shown, the pattern of CLZ_38 expression in clozapine-treated animals was similar to the pattern observed with TOGA analysis.
  • CLZ_38 mRNA expression in the brain was determined by in situ hybridization using riboprobes specific to the DST ( Figure 38A-D).
  • CLZ_38 expression was observed primarily in the white matter tracts of the brain.
  • Figure 38A,B demonstrates CLZ_38 expression in the co ⁇ us callosum (cc) and anterior commisure (ac).
  • Figure 38B demonstrates expression in the white matter of the septum (sp).
  • Figure 38C demonstrates CLZ_38 expression by cells in the hippocampal fimbria (fi).
  • Figure 38D demonstrates CLZ_38 expression in the cc, fi, and optic tract (opt).
  • CLZ_44 was up-regulated with haloperidol and ketanserin, but not clozapine. This suggests that both dopamines D2 and 5HT 2 A / 2 C receptors are involved in CLZ_44 expression regulation. The lack of effect of clozapine may indicate that antagonism at other receptors (i.e. 5HT 3 , D4, Dl) may override the effects of D2, 5HT 2 receptors.
  • in situ hybridization analysis using an antisense cRNA probe directed against the 3' end of CLZ_44 were performed to show the pattern of CLZ_44 mRNA expression in mouse anterior brain (Fig. 40A) and posterior brain (Fig. 40B).
  • Figure 40A-B is an in situ hybridization analysis using an antisense cRNA probe directed against the 3' end of CLZ_44, showing the pattern of CLZ_44 mRNA expression in a coronal section showing labelling in the hippocampus, hypothalamus, and temporal cortex (Fig. 40A) and coronal section showing cortical labelling (Fig. 40B) in mouse brain.
  • Figure 41A-B is an in situ hybridization analysis using an antisense cRNA probe directed against the 3' end of CLZ_64, showing the pattern of C Z_64 mRNA expression in different coronal sections of the hemispheres in mouse brain. As shown in Figure 41 A and B, CLZ_64 mRNA is ubiquitously expressed.
  • in situ hybridization analysis was utilized to determine the role of specific genes and their contribution to brain function. These studies demonstrate the ability of TOGA to identify genes associated with specific brain regions that could be used as tools to understand the specialized functions associated with these regions. DSTs that exhibit region specific expression could not only serve as important markers for understanding function but also drag response in the treatment of neurological disorders.
  • SEQ ID NO:l SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:ll, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO: 38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID N0:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO
  • homologues including paralogous genes and orthologous genes.
  • Homologues may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for the desired homologue.
  • polypeptides of the invention can be prepared in any suitable manner.
  • polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art. See, e.g., Curr. Prot Mol. Bio., Chapter 16.
  • polypeptides may be in the form of the secreted protein, including the mature fo ⁇ n, or may be a part of a larger protein, such as a fusion protein (see below).
  • polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified.
  • a recombinantly produced version of a polypeptide, including the secreted polypeptide, can be substantially purified by the one-step method described in Smith & Johnson (Gene, 67:31-40,
  • Polypeptides of the invention also can be purified from natural or recombinant sources using antibodies of the invention raised against the secreted protein in methods which are well known in the art.
  • the deduced amino acid sequence of the secreted polypeptide was analyzed by a computer program called Signal P (Nielsen et al, Protein Engineering, 10:1-6 (1997), which predicts the cellular location of a protein based on the amino acid sequence.
  • Signal P Neelsen et al, Protein Engineering, 10:1-6 (1997), which predicts the cellular location of a protein based on the amino acid sequence.
  • McGeoch and von Heinje are inco ⁇ orated.
  • the present invention provides secreted polypeptides having a sequence corresponding to the translations of SEQ ID NO:l, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:ll, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO: 38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:31, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:31, SEQ ID NO:40,
  • SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:68, SEQ ID NO:79, and SEQ ID NO:80 which have an N-terminus beginning within 5 residues (i.e., + or - 5 residues) of the predicted cleavage point.
  • cleavage of the signal sequence from a secreted protein is not entirely uniform, resulting in more than one secreted species.
  • These polypeptides, and the polynucleotides encoding such polypeptides are contemplated by the present invention.
  • the signal sequence identified by the above analysis may not necessarily predict the naturally occurring signal sequence.
  • the naturally occurring signal sequence may be further upstream from the predicted signal sequence.
  • the predicted signal sequence will be capable of directing the secreted protein to the ER.
  • Polynucleotide or polypeptide variants differ from the polynucleotides or polypeptides of the present invention, but retain essential properties thereof. In general, variants have close similarity overall and are identical in many regions to the polynucleotide or polypeptide of the present invention.
  • polynucleotides having at least 80% identity, more preferably at least 90% identity, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to a sequence contained in SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:ll, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO: 38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:
  • SEQ ID NO: 18 SEQ ID NO: 19, SEQ ID NO:28, SEQ ID NO 29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO 38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO 43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:68, SEQ ID NO:79, and SEQ ID NO:80.
  • polypeptides having at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence contained in translations of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:ll, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO: 38, SEQ ID NO:39, SEQ ID NO:40
  • polypeptides of the present invention include polypeptides having at least 90% similarity, more preferably at least 95%> similarity, and still more preferably at least 96%, 97%, 98%, or 99% similarity to an amino acid sequence contained in translations of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,
  • the parameters are set such that the percentage of identity is calculated over the full length of the reference polynucleotide and that gaps in identity of up to 5% of the total number of nucleotides in the reference polynucleotide are allowed.
  • a preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci, 6:237-245 (1990)).
  • sequence includes nucleotide and amino acid sequences.
  • sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences.
  • the result of said global sequence alignment is presented in terms of percent identity.
  • a polypeptide having an amino acid sequence having at least, for example, 95% "identity" to a reference polypeptide means that the amino acid sequence of the polypeptide is identical to the reference polypeptide except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the total length of the reference polypeptide.
  • up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the variants may contain alterations in the coding regions, non-coding regions, or both.
  • polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred.
  • variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also preferred.
  • Polynucleotide variants can be produced for a variety of reasons. For instance, a polynucleotide variant may be produced to optimize codon expression for a particular host (i.e., codons in the human mRNA may be changed to those preferred by a bacterial host, such as E. coli).
  • the variants may be allelic variants. Naturally occurring variants are called
  • allelic variants refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism (Lewin, Ed., Genes II, John Wiley & Sons, New York (1985)). These allelic variants can vary at either the polynucleotide and/or polypeptide level. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis. See, e.g., Curr. Prot. Mol. Bio., Chapter 8.
  • variants may be generated to improve or alter the characteristics of the polypeptides of the present invention.
  • polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as decreased aggregation.
  • aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity (see, e.g., Pinckard et al, Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al, Diabetes, 36: 838-845 (1987); Cleland et al, Crit Rev. Therap. Drug Carrier Sys., 10:307 '-377 (1993)).
  • interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein (Dobeli et al, J. Biotechnology, 7:199-216 (1988)). Moreover, ample evidence demonstrates that variants often retain a biological activity similar to that of the naturally occurring protein. For example, Gayle et al. conducted extensive mutational analysis of human cytokine IL-la (J. Biol. Chem., 268:22105-22111 (1993)). These investigators used random mutagenesis to generate over 3,500 individual IL- 1 a mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at every possible amino acid position.
  • the invention further includes polypeptide variants which show substantial biological activity.
  • Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity.
  • guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al, Science, 247:1306-1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.
  • the first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, the amino acid positions which have been conserved between species can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions in which substitutions have been tolerated by natural selection indicate positions which are not critical for protein function. Thus, positions tolerating amino acid substitution may be modified while still maintaining biological activity of the protein.
  • the second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site-directed mutagenesis or alanine-scanning mutagenesis (the introduction of single alanine mutations at every residue in the molecule) can be used (Cunningham et al, Science, 244:1081-1085 (1989)). The resulting mutant molecules can then be tested for biological activity.
  • tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and He; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gin; replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and T ⁇ ; and replacement of the small- sized amino acids Ala, Ser, Thr, Met, and Gly.
  • variants of the present invention include: (i) substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code; (ii) substitution with one or more of amino acid residues having a substituent group; (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (e.g., polyethylene glycol); (iv) fusion of the polypeptide with additional amino acids, such as an IgG Fc fusion region peptide, a leader or secretory sequence, or a sequence facilitating purification.
  • substitutions with one or more of the non-conserved amino acid residues where the substituted amino acid residues may or may not be one encoded by the genetic code
  • substitution with one or more of amino acid residues having a substituent group such as a compound to increase the stability and/or solubility of the polypeptide (e.g., poly
  • a "polynucleotide fragment” refers to a short polynucleotide having a nucleic acid sequence contained in that shown in SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:ll, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO: 38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO
  • the short nucleotide fragments are preferably at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length.
  • a fragment "at least 20 nt in length,” for example, is intended to include 20 or more contiguous bases from the cDNA sequence contained in that shown in SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:l l, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO.T5, SEQ ID NO.T6, SEQ ID NO.T7, SEQ ID NO.T8, SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO: 38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID
  • polynucleotide fragments of the invention include, for example, fragments having a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401- 450, to the end of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:ll, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO: 38,
  • a "polypeptide fragment” refers to a short amino acid sequence contained in the translations of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:ll, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO: 19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO: 38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ
  • Protein fragments may be "free-standing,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region.
  • Representative examples of polypeptide fragments of the invention include, for example, fragments from about amino acid number 1-20, 21-40, 41-60, or 61 to the end of the coding region.
  • polypeptide fragments can be about 20, 30, 40, 50, or 60 amino acids in length. In this context "about” includes the particularly recited ranges, larger or smaller by several amino acids (5, 4, 3, 2, or 1) at either extreme or at both extremes.
  • Preferred polypeptide fragments include the secreted protein as well as the mature form. Further preferred polypeptide fragments include the secreted protein or the mature form having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids ranging from 1- 60, can be deleted from the amino terminus of either the secreted polypeptide or the mature form. Similarly, any number of amino acids ranging from 1-30, can be deleted from the carboxy terminus of the secreted protein or mature form. Furthermore, any combination of the above amino and carboxy terminus deletions are preferred. Similarly, polynucleotide fragments encoding these polypeptide fragments are also preferred.
  • polypeptide and polynucleotide fragments characterized by structural or functional domains, such as fragments that comprise alpha-helix and alpha-helix-forming regions, beta-sheet and beta-sheet-forming regions, turn and turn- forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.
  • Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention.
  • the biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.
  • antigenic epitopes may be produced by any conventional means.
  • antigenic epitopes preferably contain a sequence of at least seven, more preferably at least nine, and most preferably between about 15 to about 30 amino acids.
  • Antigenic epitopes are useful to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. (See, e.g., Wilson et al, Cell, 31:161-11% (1984); Sutcliffe et al, Science, 219:660-666 (1983)).
  • immunogenic epitopes can be used to induce antibodies according to methods well known in the art. (See, e.g., Sutcliffe et al, (1983) Supra; Wilson et al, (1984) Supra; Chow et al, Proc. Natl. Acad. Sci., USA, 82:910-914; and Bittle et al, J. Gen. Virol, 66:2347-2354 (1985)).
  • a preferred immunogenic epitope includes the secreted protein.
  • the immunogenic epitope may be presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse).
  • the immunogenic epitope may be prescribed without a carrier, if the sequence is of sufficient length (at least about 25 amino acids).
  • immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting.)
  • antibody As used herein, the term "antibody” (Ab) or “monoclonal antibody” (Mab) is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab')2 fragments) which are capable of specifically binding to protein. Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al, J. Nucl. Med., 24:316-325 (1983)). Thus, these fragments are preferred, as well as the products of a Fab or other immunoglobulin expression library. Moreover, antibodies of the present invention include chimeric, single chain, and human and humanized antibodies.
  • the antibodies may be chimeric antibodies, e.g., humanized versions of murine monoclonal antibodies.
  • Such humanized antibodies may be prepared by known techniques, and offer the advantage of reduced immunogenicity when the antibodies are administered to humans. Co et al. (Nature, 351:501-2, 1991).
  • a humanized monoclonal antibody comprises the variable region of a murine antibody (or just the antigen binding site thereof) and a constant region derived from a human antibody.
  • a humanized antibody fragment may comprise the antigen binding site of a murine monoclonal antibody and a variable region fragment (lacking the antigen-binding site) derived from a human antibody.
  • One method for producing a human antibody comprises immunizing a non- human animal, such as a transgenic mouse, with a polypeptide translated from a nucleotide sequence chosen from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:l l, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO: 38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID
  • mice have been prepared in which one or more endogenous immunoglobulin genes are inactivated by various means and human immunoglobulin genes are introduced into the mice to replace the inactivated mouse genes.
  • Such transgenic mice may be genetically altered in a variety of ways. The genetic manipulation may result in human immunoglobulin polypeptide chains replacing endogenous immunoglobulin chains in at least some (preferably virtually all) antibodies produced by the animal upon immunization. Examples of techniques for production and use of such transgenic animals are described in U.S. Patent Nos. 5,814,318, 5,569,825, and 5,545,806, which are inco ⁇ orated by reference herein.
  • Monoclonal antibodies may be produced by conventional procedures, e.g., by immortalizing spleen cells harvested from the transgenic animal after completion of the immunization schedule.
  • the spleen cells may be fused with myeloma cells to produce hybridomas by conventional procedures. Examples of such techniques are described in U.S. Patent No. 4,196,265, which is inco ⁇ orated by reference herein.
  • a method for producing a hybridoma cell line comprises immunizing such a transgenic animal with an immunogen comprising at least seven contiguous amino acid residues of a polypeptide translated from a nucleotide sequence chosen from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:l l, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO: 38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ
  • hybridoma cell lines and monoclonal antibodies produced therefrom, are encompassed by the present invention.
  • Monoclonal antibodies secreted by the hybridoma cell line are purified by conventional techniques. Examples of such techniques are described in U.S. Patent No. 4,469,630 and U.S. Patent No. 4,361,549.
  • Antibodies may be employed in an in vitro procedure, or administered in vivo to inhibit biological activity induced by a polypeptide translated from a nucleotide sequence chosen from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:ll, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:37, S rEQ ID NO: 38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:
  • disorders caused or exacerbated (directly or indirectly) by the interaction of such polypeptides of the present invention with cell surface receptors thus may be treated.
  • chronic administration of neuroleptics can cause unwanted side effects.
  • Administration of an antibody derived from the identified polynucleotides might block the signaling that causes these side effects.
  • an antibody derived from the identified polynucleotides might selectively block proteins causing motor side effects.
  • a therapeutic method involves in vivo administration of a blocking antibody to a mammal in an amount effective for reducing a biological activity induced by a polypeptide translated from a nucleotide sequence chosen from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:ll, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID N0:31, SEQ ID NO:36, SEQ ID NO:
  • conjugates comprising a detectable (e.g., diagnostic) or therapeutic agent, attached to an antibody directed against a polypeptide translated from a nucleotide sequence chosen from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO.IO, SEQ ID NO:ll, SEQ ID N0.12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO: 38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:
  • Such agents include but are not limited to diagnostic radionuclides, therapeutic radionuclides, and cytotoxic drugs. See, e.g., Thrush et. al (Annu.Rev.ImmunoL, 14:49-71, 1996, p. 41).
  • the conjugates find use in in vitro or in vivo procedures.
  • any polypeptide of the present invention can be used to generate fusion proteins.
  • the polypeptide of the present invention when fused to a second protein, can be used as an antigenic tag.
  • Antibodies raised against the polypeptide of the present invention can be used to indirectly detect the second protein by binding to the polypeptide.
  • the polypeptides of the present invention can be used as targeting molecules once fused to other proteins. Examples of domains that can be fused to polypeptides of the present invention include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily need to be direct, but may occur through linker sequences.
  • fusion proteins may also be engineered to improve characteristics of the polypeptide of the present invention. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence during purification from the host cell or subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to facilitate handling of polypeptides are familiar and routine techniques in the art.
  • polypeptides of the present invention can be combined with parts of the constant domain of immunoglobulms (IgG), resulting in chimeric polypeptides.
  • IgG immunoglobulms
  • EP A 0 464 533 (Canadian counte ⁇ art 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof.
  • the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties (see, e.g., EP A 0 232 262).
  • deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired.
  • the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations.
  • human proteins such as hIL-5
  • Fc portions for the pu ⁇ ose of high-throughput screening assays to identify antagonists of hIL-5 (See, Bennett et al, J. Mol. Recognition 8:52-58 (1995); Johanson et al, J. Biol.
  • the polypeptides of the present invention can be fused to marker sequences, such as a peptide which facilitates purification of the fused polypeptide.
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., Chatsworth, CA), among others, many of which are commercially available.
  • hexa-histidine provides for convenient purification of the fusion protein (Proc. Natl. Acad. Sci. USA 86:821-824 (1989)).
  • HA hemagglutinin protein
  • Other fusion proteins may use the ability of the polypeptides of the present invention to target the delivery of a biologically active peptide. This might include focused delivery of a toxin to tumor cells, or a growth factor to stem cells.
  • any of these above fusions can be engineered using the polynucleotides or the polypeptides of the present invention. See, e.g., Curr. Prot. Mol. Bio., Chapter 9.6.
  • the present invention also relates to vectors containing the polynucleotide of the present invention, host cells, and the production of polypeptides by recombinant techniques.
  • the vector may be, for example, a phage, plasmid, viral, or retroviral vector.
  • Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
  • the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host.
  • a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid.
  • the vector may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells. See, e.g., Curr. Prot. Mol. Bio., Chapters 9.9, 16.15.
  • the polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, tip, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be .known * to the skilled artisan.
  • the expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
  • the expression vectors will preferably include at least one selectable marker.
  • markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria.
  • Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells, and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
  • vectors preferred for use in bacteria include pQ ⁇ 70, pQE60 and pQE- 9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, PNH16A, PNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc.
  • eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
  • Other suitable vectors will be readily apparent to the skilled artisan.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al, Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may, in fact, be expressed by a host cell lacking a recombinant vector.
  • a polypeptide of this invention can be recovered and purified from recombinant cell cultures by well-known methods, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Most preferably, high performance liquid cl romatography (“HPLC”) is employed for purification.
  • HPLC high performance liquid cl romatography
  • polypeptides of the present invention may be glycosylated or may be non- glycosylated.
  • polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
  • N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.
  • Polypeptides of the present invention can also be recovered from products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells.
  • a polynucleotdie of the invention is up-regulated, such as after chronic treatment with clozapine
  • the expression of the polynucleotide can be increased or the level of the intact polypeptide product can be increased in order to treat, prevent, ameliorate, or modulate the pathological condition.
  • increased expression of the SEQ ID NO:# (CLZ_5) was observed after chronic treatment with clozapine.
  • a polynucleotide of the invention can be administered alone or withother polynucleotides to a mammalian subject by a recombinant expression vector comprising the polynucleotide.
  • a mammalian subject can be a human, baboon, chimpanzee, macaque, cow, horse, sheep, pig, horse, dog, cat, rabbit, guinea pig, rat or mouse.
  • the recombinant vector comprises a polynucleotide shown in SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO.ll, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO: 38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID N0.41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO
  • the recombinant vector comprises a variant polynucleotide that is at least 80%, 90%, or 95% identical to a polynucleotide comprising SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO.l l, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO: 38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:40,
  • a polynucleotide or recombinant expression vector of the invention can be used to express a polynucleotide in said subject for the treatment of neurological and psychiatric disorders, for example, schizophrenia.
  • Expression of a polynucleotide in target cells including but not limited to brain cells, would effect greater production of the encoded polypeptide.
  • the regulation of other genes may be secondarily up- or down-regulated.
  • a polynucleotide or recombinant expression vector of the invention can be used to express a polynucleotide in the said subject for the treatment of, for example, psychosis or other neuropsychiatric disorders.
  • Expression of a polynucleotide in target cells including but not limited to the cells of the striatum and nucleus accumbens, would effect greater production of the encoded polypeptide.
  • a naked polynucleotide can be administered to target cells.
  • Polynucleotides and recombinant expression vectors of the invention can be administered as a pharmaceutical composition.
  • Such a composition comprises an effective amount of a polynucleotide or recombinant expression vector, and a pharmaceutically acceptable formulation agent selected for suitability with the mode of administration.
  • Suitable formulation materials preferably are non-toxic to recipients at the concentrations employed and can modify, maintain, or preserve, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adso ⁇ tion, or penetration of the composition. See Remington 's Pharmaceutical Sciences (18th Ed., A.R. Gennaro, ed., Mack Publishing Company 1990).
  • the pharmaceutically active compounds i.e., a polynucleotide or a vector
  • the pharmaceutical composition comprising a polynucleotide or a recombinant expression vector may be made up in a solid form (including granules, powders or suppositories) or in a liquid form (e.g., solutions, suspensions, or emulsions).
  • the dosage regimen for treating a disease with a composition comprising a polynucleotide or expression vector is based on a variety of factors, including the type or severity of the neurological or psychiatric disorder the age, weight, sex, medical condition of the patient, the route of administration, and the particular compound employed. Thus, the dosage regimen may vary widely, but can be determined routinely using standard methods. A typical dosage may range from about 0.1 mg/kg to about 100 mg/kg or more, depending on the factors mentioned above.
  • the frequency of dosing will depend upon the pharmacokinetic parameters of the polynucleotide or vector in the formulation being used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect.
  • composition may therefore be administered as a single dose, as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages may be ascertained through use of appropriate dose-response data.
  • the cells of a mammalian subject may be transfected in vivo, ex vivo, or in vitro.
  • Administration of a polynucleotide or a recombinant vector containing a polynucleotide to a target cell in vivo may be accomplished using any of a variety of techniques well known to those skilled in the art.
  • U.S. Patent No. 5,672,344 describes an in vivo viral-mediated gene transfer system involving a recombinant neurotrophic HSV-1 vector.
  • compositions of' polynucleotides and recombinant vectors can be transfected in vivo by oral, buccal, parenteral, rectal, or topical administration as well as by inhalation spray.
  • parenteral as used herein includes, subcutaneous, intravenous, intramuscular, intrasternal, infusion techniques or intraperitoneally.
  • nucleic acids and/or vectors of the invention can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more vectors of the invention or other agents.
  • the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.
  • Another delivery system for polynucleotides of the invention is a "non- viral" delivery system.
  • Techniques that have been used or proposed for gene therapy include DNA-ligand complexes, adenoviras-ligand-DNA complexes, direct injection of DNA, CaPO4 precipitation, gene gun techniques, electroporation, lipofection, and colloidal dispersion (Mulligan, R., (1993) Science, 260 (5110):926-32). Any of these methods are widely available to one skilled in the art and would be suitable for use in the present invention. Other suitable methods are available to one skilled in the art, and it is to be understood that the present invention may be accomplished using any of the available methods of transfection. Several such methodologies have been utilized by those skilled in the art with varying success (Mulligan, R., (1993) Science, 260
  • a polynucleotide of the invention is down-regulated, such as after chronic treatment with clozapine, the expression of the polynucleotide can be blocked or reduced or the level of the intact polypeptide product can be reduced in order to treat, prevent, ameliorate, or modulate the pathological condition, such as psychosis or other neuropsychiatric disorders.
  • the pathological condition such as psychosis or other neuropsychiatric disorders.
  • decreased expression of the polynucleotides with the SEQ ID NOs: 15, 28, 29, and 12 were down-regulated after chronic administration of clozapine. This activity may represent pathways common to the beneficial effects of clozapine treatment of psychosis or other neuropsychiatric disorders.
  • Antisense oligonucleotides are nucleotide sequences which are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation.
  • an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used.
  • Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of gene products of the invention in the cell.
  • Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester intemucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown, (1994) Meth. Mol. Biol, 20: 1-8; Sonveaux, (1994) Meth. Mol. Biol, 26: 1-72; Uhlmann et al, (1990) Chem. Rev., 90:543-583.
  • Modifications of gene expression can be obtained by designing antisense oligonucleotides which will form duplexes to the control, 5', or regulatory regions of a gene of the invention. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred.
  • triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons.
  • Therapeutic advances using triplex DNA have been described in the literature (e.g., Gee et al, in Huber & Carr, MOLECULAR AND IMMUNOLOGIC APPROACHES, Futura Publishing Co., Mt. Kisco, N.Y., 1994).
  • An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent nucleotides, can provide sufficient targeting specificity for mRNA.
  • each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 1, or 8 or more nucleotides in length.
  • Non-complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length.
  • One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular polynucleotide sequence.
  • Antisense oligonucleotides can be modified without affecting their ability to hybridize to a polynucleotide of the invention. These modifications can be internal or at one or both ends of the antisense molecule.
  • internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose.
  • Modified bases and/or sugars such as arabinose instead of ribose, or a 3', 5'- substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide.
  • modified oligonucleotides can be prepared by methods well known in the art. See, e.g., Agrawal et al, (1992) Trends Biotechnol, 10:152-158; Uhlmann et al, (1990) Chem. Rev., 90:543-584; Uhlmann et al, (1987) Tetrahedron. Lett, 215:3539-3542.
  • Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, (1987)
  • Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al, U.S. Patent 5,641,673).
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.
  • the coding sequence of a polynucleotide of the invention can be used to generate ribozymes which will specifically bind to mRNA transcribed from the polynucleotide.
  • Methods of designing and constructing ribozymes which can cleave RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al. (1988) Nature, 334:585-591).
  • the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme.
  • the hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, e.g., Gerlach et al, EP 321,201).
  • Specific ribozyme cleavage sites within a RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
  • SEQ ID NO:l The nucleotide sequences shown in SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l l, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID N0:18, SEQ ID N0:19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID N0:31, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO: 38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID N0:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, S
  • hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.
  • Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease polynucleotide expression. Alternatively, ifit is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art.
  • a ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells.
  • ribozymes can be engineered so that ribozyme expression will occur in response to factors which induce expression of a target gene. Ribozymes also can be engineered to provide an additional level of regulation, so that destraction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells.
  • Pathological conditions or susceptibility to pathological conditions can be diagnosed using methods of the invention. Testing for expression of a polynucleotide of the invention or for the presence of the polynucleotide product can correlate with the severity of the condition and can also indicate appropriate treatment. For example, the presence or absence of a mutation in a polynucleotide of the invention can be determined and a pathological condition or a susceptibility to a pathological condition is diagnosed based on the presence or absence of the mutation.
  • an alteration in expression of a polypeptide encoded by a polynucleotide of the invention can be detected, where the presence of an alteration in expression of the polypeptide is indicative of the pathological condition or susceptibility to the pathological condition.
  • the alteration in expression can be an increase in the amount of expression or a decrease in the amount of expression.
  • a first biological sample from a patient suspected of having a pathological condition is obtained along with a second sample from a suitable comparable control source.
  • a biological sample can comprise saliva, blood, cerebrospinal fluid, amniotic fluid, urine, feces, or tissue, such as gastrointestinal tissue.
  • a suitable control source can be obtained from one or more mammalian subjects that do not have the pathological condition.
  • the average concentrations and distribution of a polynucleotide or polypeptide of the invention can be determined from biological samples taken from a representative population of mammalian subjects, wherein the mammalian subjects are the same species as the subject from which the test sample was obtained.
  • the amount of at least one polypeptide encoded by a polynucleotide of the invention is determined in the first and second sample.
  • the amounts of the polypeptide in the first and second samples are compared.
  • a patient is diagnosed as having a pathological condition if the amount of the polypeptide in the first sample is greater than or less than the amount of the polypeptide in the second sample.
  • the amount of polypeptide in the first sample falls within the range of samples taken from a representative group of patients with the pathological condition.
  • the method for diagnosing a pathological condition can comprise a step of detecting nucleic acid molecules comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from said group.
  • the present invention also includes a diagnostic system, preferably in kit form, for assaying for the presence of the polypeptide of the present invention in a body sample, such as brain tissue, cell suspensions or tissue sections; or a body fluid sample, such as CSF, blood, plasma or serum, where it is desirable to detect the presence, and preferably the amount, of the polypeptide of this invention in the sample according to the diagnostic methods described herein.
  • a body sample such as brain tissue, cell suspensions or tissue sections
  • a body fluid sample such as CSF, blood, plasma or serum
  • a nucleic acid molecule can be used as a probe (i.e., an oligonucleotide) to detect the presence of a polynucleotide of the present invention, a gene corresponding to a polynucleotide of the present invention, or a mRNA in a cell that is diagnostic for the presence or expression of a polypeptide of the present invention in the cell.
  • the nucleic acid molecule probes can be of a variety of lengths from at least about 10, suitably about 10 to about 5000 nucleotides long, although they will typically be about 20 to 500 nucleotides in length.
  • the probe can be used to detect the polynucleotide through] hybridization methods which are extremely well known in the art and will not be described further here.
  • PCR primers are utilized in pairs, as is well known, based on the nucleotide sequence of the gene to be detected.
  • the nucleotide sequence is a portion of the nucleotide sequence of a polynucleotide of the present invention.
  • Particularly preferred PCR primers can be derived from any portion of a DNA sequence encoding a polypeptide of the present invention, but are preferentially from regions which are not conserved in other cellular proteins.
  • PCR primer pairs useful for detecting the genes corresponding to the polynucleotides of the present invention and expression of these genes are described in the TOGA TM Process Section above and in the Tables. Nucleotide primers from the corresponding region of the polypeptides of the present invention described herein are readily prepared and used as PCR primers for detection of the presence or expression of the corresponding gene in any of a variety of tissues.
  • a diagnostic system preferably in kit form, is contemplated for assaying for the presence of the polypeptide of the present invention or an antibody immunoreactive with the polypeptide of the present invention in a body fluid sample.
  • Such diagnostic kit would be useful for monitoring the fate of a therapeutically administered polypeptide of the present invention or an antibody immunoreactive with the polypeptide of the present invention.
  • the system includes, in an amount sufficient for at least one assay, a polypeptide of the present invention and/or a subject antibody as a separately packaged immunochemical reagent. Instructions for use of the packaged reagent(s) are also typically included.
  • a diagnostic system of the present invention preferably also includes a label or indicating means capable of signaling the formation of an immunocomplex containing a polypeptide or antibody molecule of the present invention.
  • Any label or indicating means can be linked to or inco ⁇ orated in an expressed protein, polypeptide, or antibody molecule that is part of an antibody or monoclonal antibody composition of the present invention or used separately, and those atoms or molecules can be used alone or in conjunction with additional reagents.
  • Such labels are themselves well-known in clinical diagnostic chemistry and constitute a part of this invention only insofar as they are utilized with otherwise novel proteins methods and/or systems.
  • the labeling means can be a fluorescent labeling agent that chemically binds to antibodies or antigens without denaturing them to form a fluorochrome (dye) that is a useful immunofluorescent tracer.
  • Suitable fluorescent labeling agents are fluorochromes such as fluorescein isocyanate (FIC), fluorescein isothiocyanate (FITC), 5 -dimethylamine- 1-naphthalenesulfonyl chloride (DANSC), tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200 sulphonyl chloride (RB 200 SC) and the like.
  • fluorochromes such as fluorescein isocyanate (FIC), fluorescein isothiocyanate (FITC), 5 -dimethylamine- 1-naphthalenesulfonyl chloride (DANSC), tetramethylrhodamine isothiocyanate (TRI
  • the indicating group is an enzyme, such as horseradish peroxidase (HRP), glucose oxidase, or the like.
  • HRP horseradish peroxidase
  • glucose oxidase or the like.
  • additional reagents are required to visualize the fact that a receptor-ligand complex (immunoreactant) has formed.
  • additional reagents for HRP include hydrogen peroxide and an oxidation dye precursor such as diaminobenzidine.
  • An additional reagent useful with glucose oxidase is 2,2'-amino-di-(3-ethyl-benzthiazoline-G- sulfonic acid) (ABTS).
  • Radioactive elements are also useful labeling agents and are used illustratively herein.
  • An exemplary radiolabeling agent is a radioactive element that produces gamma ray emissions. Elements which themselves emit gamma rays, such as 12 1,
  • I, I, l and Cr represent one class of gamma ray emission-producing radioactive element indicating groups. Particularly preferred is 125 I.
  • Another group of useful labeling means are those elements such as ⁇ C, 18 F, 15 O and 13 N which themselves emit positrons. The positrons so emitted produce gamma rays upon encounters with electrons present in the animal's body.
  • a beta emitter such 11 indium or 3 H.
  • antibody molecules produced by a hybridoma can be labeled by metabolic inco ⁇ oration of radioisotope-containing amino acids provided as a component in the culture medium (see, e.g., Galfre et al, Meth. Enzymol, 73:3-46 (1981)).
  • the techniques of protein conjugation or coupling through activated functional groups are particularly applicable (see, e.g., Aurameas, et al, Scand. J. Immunol, Vol. 8 Suppl. 7:7-23 (1978); Rodwell et al, Biotech., 3:889-894 (1984); and U.S. Patent No. 4,493,795).
  • the diagnostic systems can also include, preferably as a separate package, a specific binding agent.
  • exemplary specific binding agents are second antibody molecules, complement proteins or fragments thereof, S. aureus protein A, and the like.
  • the specific binding agent binds the reagent species when that species is present as part of a complex.
  • the specific binding agent is labeled.
  • the agent is typically used as an amplifying means or reagent.
  • the labeled specific binding agent is capable of specifically binding the amplifying means when the amplifying means is bound to a reagent species-containing complex.
  • the diagnostic kits of the present invention can be used in an "ELISA" format to detect the quantity of the polypeptide of the present invention in a sample.
  • ELISA ELISA
  • a description of the ELISA technique is found in Sites et al, Basic and Clinical Immunology, 4th Ed., Chap. 22, Lange Medical Publications, Los Altos, CA (1982) and in U.S. Patent No. 3,654,090; U.S. Patent No. 3,850,752; and U.S. Patent No. 4,016,043, which are all inco ⁇ orated herein by reference.
  • a polypeptide of the present invention an antibody or a monoclonal antibody of the present invention can be affixed to a solid matrix to form a solid support that comprises a package in the subject diagnostic systems.
  • a reagent is typically affixed to a solid matrix by adso ⁇ tion from an aqueous medium, although other modes of affixation applicable to proteins and polypeptides can be used that are well known to those skilled in the art. Exemplary adso ⁇ tion methods are described herein.
  • Useful solid matrices are also well known in the art.
  • Such materials are water insoluble and include the cross-linked dextran available under the trademark SEPHADEX from Pharmacia Fine Chemicals (Piscataway, NJ), agarose, polystyrene beads of about 1 micron ( ⁇ m) to about 5 millimeters (mm) in diameter available from several suppliers (e.g., Abbott Laboratories, Chicago, IL), polyvinyl chloride, polystyrene, cross-linked polyacrylamide, nitrocellulose- or nylon-based webs (sheets, strips or paddles) or tubes, plates or the wells of a microtiter plate, such as those made from polystyrene or polyvinylchloride.
  • SEPHADEX Pharmacia Fine Chemicals
  • agarose agarose
  • polystyrene beads of about 1 micron ( ⁇ m) to about 5 millimeters (mm) in diameter available from several suppliers (e.g., Abbott Laboratories, Chicago, IL)
  • polyvinyl chloride polystyrene
  • the reagent species, labeled specific binding agent, or amplifying reagent of any diagnostic system described herein can be provided in solution, as a liquid dispersion or as a substantially dry power, e.g., in lyophilized form.
  • the indicating means is an enzyme
  • the enzyme's substrate can also be provided in a separate package of a system.
  • a solid support such as the before-described microtiter plate and one or more buffers can also be included as separately packaged elements in this diagnostic assay system.
  • the packaging materials discussed herein in relation to diagnostic systems are those customarily utilized in diagnostic systems.
  • the polynucleotides of the present invention are useful for chromosome identification. There exists an ongoing need to identify new chromosome markers, since few chromosome marking reagents based on actual sequence data (repeat polymo ⁇ hisms) are presently available. Each polynucleotide of the present invention can be used as a chromosome marker. Currently, no specific diagnostic markers exist that can be used to prevent or delay psychotic episodes of schizophrenia. The polynucleotides of the present invention may be used as chromosome markers for diagnosis for schizophrenia.
  • sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the sequences shown in SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:ll, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO: 38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID
  • Primers can be selected using computer analysis so that primers do not span more than one predicted exon in the genomic DNA. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:l l, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO: 19, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO: 38, SEQ ID NO:39, SEQ ID NO:
  • somatic hybrids provide a rapid method of PCR mapping the polynucleotides to particular chromosomes. Three or more clones can be assigned per day using a single thermal cycler. Moreover, sublocalization of the polynucleotides can be achieved with panels of specific chromosome fragments.
  • Other gene-mapping strategies that can be used include in situ hybridization, prescreening with labeled flow-sorted chromosomes, and preselection by hybridization to construct chromosome specific-cDNA libraries.
  • FISH fluorescence in situ hybridization
  • the polynucleotides can be used individually (to mark a single chromosome or a single site on that chromosome) or in panels (for marking multiple sites and/or multiple chromosomes).
  • Preferred polynucleotides correspond to the noncoding regions of the cDNAs because the coding sequences are more likely conserved within gene families, thus increasing the chance of cross- hybridization during chromosomal mapping.
  • Linkage analysis establishes coinheritance between a chromosomal location and presentation of a particular disease.
  • Disease mapping data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library), Kruglyak et al. (Am. J. Hum. Genet, 56:1212-23, 1995); Curr. Prot. Hum. Genet Assuming one megabase mapping resolution and one gene per 20 kb, a cDNA precisely localized to a chromosomal region associated with the disease could be one of 50-500 potential causative genes.
  • polynucleotide and the corresponding gene between affected and unaffected individuals can be examined.
  • a polynucleotide can be used to control gene expression through triple helix formation or antisense DNA or RNA. Both methods rely on binding of the polynucleotide to DNA or RNA.
  • preferred polynucleotides are usually 20 to 40 bases in length and complementary to either the region of the gene involved in transcription (see, Lee et al, Nuc Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervan et al, Science, 251:1360 (1991) for discussion of triple helix formation) or to the mRNA itself (see, Okano, J Neurochem, 56:560 (1991); and Oligodeoxy-nucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988) for a discussion of antisense technique).
  • Triple helix formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques are effective in model systems, and the information disclosed herein can be used to design antisense or triple helix polynucleotides in an effort to treat disease.
  • a polypeptide of the present invention can be used to assay protein levels in a biological sample using antibody-based techniques.
  • protein expression in tissues can be studied with classical immunohistological methods (Jalkanen, et al, J. Cell. Biol, 101:976-985 (1985); Jalkanen, et al., J. Cell. Biol, 105:3087-3096 (1987)).
  • Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). See, e.g., Curr. Prot.
  • Suitable antibody assay labels include enzyme labels, such as glucose oxidase; and radioisotopes, such as iodine ( 125 1, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( ⁇ ⁇ In), and technetium ( 99m Tc); fluorescent labels, such as fluorescein and rhodamine; and biotin.
  • enzyme labels such as glucose oxidase
  • radioisotopes such as iodine ( 125 1, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( ⁇ ⁇ In), and technetium ( 99m Tc)
  • fluorescent labels such as fluorescein and rhodamine
  • biotin such as fluorescein and rhodamine
  • proteins can also be detected in vivo by imaging.
  • Antibody labels or markers for in vivo imaging of protein include those detectable by X-radiography, nuclear magnetic resonance (NMR), or electron spin resonance (ESR).
  • suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject.
  • suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be inco ⁇ orated into the antibody by labeling of nutrients for the relevant hybridoma.
  • a protein-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety such as a radioisotope (e.g., 131 I, m In, 99m Tc), a radio-opaque substance, or a material detectable by NMR, is introduced (e.g., parenterally, subcutaneously, or intraperitoneally) into the mammal.
  • a radioisotope e.g., 131 I, m In, 99m Tc
  • a radio-opaque substance e.g., a radio-opaque substance, or a material detectable by NMR
  • the labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein.
  • In vivo tumor imaging is described in Burchiel et al, "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments" (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, Burchiel and Rhodes, Eds., Masson Publishing Inc. (1982)).
  • the invention provides a diagnostic method of a disorder, which involves (a) assaying the expression of a polypeptide of the present invention in cells or body fluid of an individual; and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a disorder.
  • Psychiatric disorders and treatment of psychiatric disorders with neuroleptics, including schizophrenia are associated with a dysregulation of neurotransmitter and/or neuropeptide levels that can result in the up- or down regulation of polynucleotides and polypeptides. These changes can be diagnosed or monitored by assaying changes in polypeptide levels in tissue or fluids such as CSF, blook, or in fecal samples.
  • polypeptides of the present invention can be used to treat disease.
  • schizophrenic patients can be administered a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide; to supplement absent or decreased levels of a different polypeptide (e.g., hemoglobin S for hemoglobin B); to inhibit the activity of a polypeptide (e.g., an oncogene); to activate the activity of a polypeptide (e.g., by binding to a receptor); to reduce the activity of a membrane bound receptor by competing with it for free ligand (e.g., soluble tumor necrosis factor (TNF) receptors used in reducing inflammation); or to bring about a desired response (e.g., blood vessel growth).
  • free ligand e.g., soluble tumor necrosis factor (TNF) receptors used in reducing inflammation
  • antibodies directed to a polypeptide of the present invention can also be used to treat disease.
  • administration of an antibody directed to a polypeptide of the present invention can bind and reduce ove ⁇ roduction of the polypeptide.
  • administration of an antibody can activate the polypeptide, such as by binding to a polypeptide bound to a membrane (receptor).
  • Polypeptides can be used as antigens to trigger immune responses. Local production of neurotransmitters and neuropeptides modulates many aspects of neuronal function. For example, in schizophrenia overactive neurotransmitter activity is thought to be basis for the psychotic behavior.
  • Administration of an antibody to an ove ⁇ roduced polypeptide can be used to modulate neuronal responses in psychiatric disorders such as schizophrenia.
  • Polypeptides can also be used to raise antibodies, which in rum are used to measure protein expression from a recombinant cell, as a way of assessing transformation of the host cell. See, e.g., Curr. Prot. Mol. Bio., Chapterl 1.15. Moreover, the polypeptides of the present invention can be used to test the following biological activities.
  • polynucleotides and polypeptides of the present invention can be used in assays to test for one or more biological activities. If these polynucleotides and polypeptides do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides and polypeptides could be used to treat the associated disease.
  • a polypeptide or polynucleotide of the present invention may be useful in treating deficiencies or disorders of the central nervous system or peripheral nervous system by activating or inliibiting the proliferation, differentiation, or mobilization (chemotaxis) of neuroblasts, stem cells, or glial cells. Also, a polypeptide or polynucleotide of the present invention may be useful in treating deficiencies or disorders of the central nervous system or peripheral nervous system by activating or inhibiting the mechanisms of synaptic transmission, synthesis, metabolism and inactivation of neural transmitters, neuromodulators and trophic factors, and by activating or inhibiting the expression and inco ⁇ oration of enzymes, structural proteins, membrane channels, receptors in neurons and glial cells, or altering neural membrane compositions.
  • etiology of these deficiencies or disorders may be genetic, somatic (such as cancer or some autoimmune disorder), acquired (e.g., by chemotherapy or toxins), or infectious.
  • a polynucleotide or polypeptide of the present invention can be used as a marker or detector of a particular nervous system disease or disorder.
  • the disorder or disease can be any of Alzheimer's disease, Pick's disease,
  • Binswanger's disease other senile dementia, Parkinson's disease, parkinsonism, obsessive compulsive disorders, epilepsy, encephaolopathy, ischemia, alcohol addiction, drag addiction, schizophrenia, amyotrophic lateral sclerosis, multiple sclerosis, depression, and bipolar manic-depressive disorder.
  • the polypeptide or polynucleotide of the present invention can be used to study circadian variation, aging, or long-term potentiation, the latter affecting the hippocampus.
  • the polypeptide or polynucleotide of the present invention can be used to study brain regions that are known to be involved in complex behaviors, such as learning and memory, emotion, drug addiction, glutamate neurotoxicity, feeding behavior, olfaction, viral infection, vision, and movement disorders. Binding Activity
  • a polypeptide of the present invention may be used to screen for molecules that bind to the polypeptide or for molecules to which the polypeptide binds.
  • the binding of the polypeptide and the molecule may activate (i.e., an agonist), increase, inhibit (i.e., an antagonist), or decrease activity of the polypeptide or the molecule bound.
  • Examples of such molecules include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.
  • the molecule is closely related to the natural ligand of the polypeptide, e.g., a fragment of the ligand, or a natural substrate, a ligand, a structural or functional mimetic (see, Coligan et al, Current Protocols in Immunology 1(2), Chapter 5 (1991)).
  • the molecule can be closely related to the natural receptor to which the polypeptide binds or, at least, related to a fragment of the receptor capable of being bound by the polypeptide (e.g., an active site).
  • the molecule can be rationally designed using known techniques.
  • the screening for these molecules involves producing appropriate cells which express the polypeptide, either as a secreted protein or on the cell membrane.
  • Preferred cells include cells from mammals, yeast, Drosophila, or E. coli.
  • Cells expressing the polypeptide (or cell membrane containing the expressed polypeptide) are then preferably contacted with a test compound potentially containing the molecule to observe binding, stimulation, or inhibition of activity of either the polypeptide or the molecule.
  • the assay may simply test binding of a candidate compound to the polypeptide, wherein binding is detected by a label, or in an assay involving competition with a labeled competitor. Further, the assay may test whether the candidate compound results in a signal generated by binding to the polypeptide. Alternatively, the assay can be carried out using cell-free preparations, polypeptide/molecule affixed to a solid support, chemical libraries, or natural product mixtures. The assay may also simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide, measuring polypeptide/molecule activity or binding, and comparing the polypeptide/molecule activity or binding to a standard.
  • an ELISA assay can measure polypeptide level or activity in a sample (e.g., biological sample) using a monoclonal or polyclonal antibody.
  • the antibody can measure polypeptide level or activity by either binding, directly or indirectly, to the polypeptide or by competing with the polypeptide for a substrate.
  • the molecules discovered using these assays can be used to treat disease or to bring about a particular result in a patient (e.g., blood vessel growth) by activating or inhibiting the polypeptide/molecule.
  • the assays can discover agents which may inhibit or enhance the production of the polypeptide from suitably manipulated cells or tissues.
  • diagnosis of schizophrenia is based on clinical assessment and not on any lab test. Thus, the availability of an objective laboratory diagnostic will be of great value in the diagnosis and assessment of patients through treatment regimens.
  • the invention includes a method of identifying compounds which bind to a polypeptide of the invention comprising the steps of: (a) incubating a candidate binding compound with a polypeptide of the invention; and (b) determining if binding has occurred.
  • the invention includes a method of identifying agonists/antagonists comprising the steps of: (a) incubating a candidate compound with a polypeptide of the invention, (b) assaying a biological activity, and (c) determining if a biological activity of the polypeptide has been altered.
  • a polypeptide or polynucleotide of the present invention may also increase or decrease the differentiation or proliferation of embryonic stem cells from a lineage other than the above-described hemopoietic lineage.
  • a polypeptide or polynucleotide of the present invention may also increase or decrease the differentiation or proliferation of embryonic stem cells from a lineage other than the above-described hemopoietic lineage.
  • Expression of a polynucleotide or polypeptide of the present invention may be associated with various types of CNS pathology, including psychosis or other neuropsychiatric disorders.
  • SEQ ID NO: 2 (CLZ_5) expression has been associated not only been associate with clozapine treatment, and schizophrenic and bipolar patients, it has also been associated with specific brain regions of a mouse model for Alzheimer's disease.
  • Alzheimer's disease is an example of a disease that is accompanied by degenerating neuronal cells. Repopulation of lost neurons would be a feasible treatment option if molecules existed to promote the differentiation.
  • a polypeptide or polynucleotide of the present invention may also be used to modulate mammalian characteristics, such as body height, weight, hair color, eye color, skin, percentage of adipose tissue, pigmentation, size, and shape (e.g., cosmetic surgery).
  • a polypeptide or polynucleotide of the present invention may be used to modulate mammalian metabolism affecting catabolism, anabolism, processing, utilization, and storage of energy.
  • a polypeptide or polynucleotide of the present invention may be used to change a mammal's mental state or physical state by influencing biorhythms, circadian rhythms, depression (including depressive disorders), tendency for violence, tolerance for pain, the response to opiates and opioids, tolerance to opiates and opioids, withdrawal from opiates and opioids, reproductive capabilities (preferably by activin or inhibin-like activity), hormonal or endocrine levels, appetite, libido, memory, stress, or other cognitive qualities.
  • a polypeptide or polynucleotide of the present invention may also be used as a food additive or preservative, such as to increase or decrease storage capabilities, fat content, lipid, protein, carbohydrate, vitamins, minerals, cofactors, or other nutritional components.
  • ApoD concentrations were measured by ELISA using purified apoD as a standard.
  • CLZ_3 Serine protease Cortex, Thalamus, Hippocampus, Striatum, Amygdala

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Abstract

L'invention concerne des polynucléotides, des polypeptides, des trousses et des méthodes qui sont liées à des gènes exprimés dans le système nerveux central, lesquels sont régulés par des neuroleptiques.
PCT/US2001/030695 2000-09-29 2001-10-01 Expression genetique dans le systeme nerveux central regule par des neuroleptiques WO2002026936A2 (fr)

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AU2001296451A AU2001296451A1 (en) 2000-09-29 2001-10-01 Gene expression in the central nervous system regulated by neuroleptic agents
AU2001296451A AU2001296451A8 (en) 2000-09-29 2001-10-01 Gene expression in the central nervous system regulated by neuroleptic agents
US10/381,957 US20070010664A1 (en) 2001-10-01 2001-10-01 Gene expression in the central nervous system regulated by neuroleptic agents

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7736654B2 (en) 2001-04-10 2010-06-15 Agensys, Inc. Nucleic acids and corresponding proteins useful in the detection and treatment of various cancers

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7736654B2 (en) 2001-04-10 2010-06-15 Agensys, Inc. Nucleic acids and corresponding proteins useful in the detection and treatment of various cancers

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