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WO2004092417A2 - Molecular correlates of cocaine addiction and methods for their use - Google Patents

Molecular correlates of cocaine addiction and methods for their use Download PDF

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Publication number
WO2004092417A2
WO2004092417A2 PCT/US2004/010649 US2004010649W WO2004092417A2 WO 2004092417 A2 WO2004092417 A2 WO 2004092417A2 US 2004010649 W US2004010649 W US 2004010649W WO 2004092417 A2 WO2004092417 A2 WO 2004092417A2
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Prior art keywords
genes
expression
addictive
levels
cocaine
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PCT/US2004/010649
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French (fr)
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WO2004092417A3 (en
Inventor
Scott Edwards Hemby
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Emory University
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Publication of WO2004092417A3 publication Critical patent/WO2004092417A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates generally to molecular correlates of drug addiction and more specifically to the identification of molecular targets for use in diagnosing and treating addictive disorders.
  • the present invention provides methods for identifying genes and their expression products as targets in the diagnosis and treatment of addictive disorders.
  • the targets are identified based on their differential expression in subjects affected by an addictive disorder in comparison with control subjects.
  • Genes whose expression level is significantly increased or significantly decreased in subjects affected by an addictive disorder in comparison with control subjects are identified, along with their expression products, as screening targets for use in methods of identifying drugs for treating addictive disorders.
  • the invention provides an improved method for screening for therapeutic compounds for use in treating addictive disorders, where the improvement comprises the use of a target identified by the method described above.
  • the present invention provides methods for producing an expression profile having values representing the expression levels of genes whose expression is correlated with addictive disorders. The method comprises determining the expression level of one or more genes in a sample from subjects affected by an addictive disorder, determining the expression levels of the same genes in control subjects, and identifying genes whose expression level is significantly increased or significantly decreased in subjects affected by the addictive disorder in comparison with control subjects.
  • the present invention also provides methods for predicting whether a compound will be addictive in humans.
  • the methods comprise the steps of providing a reference expression profile associated with an addictive disorder, where the expression profile comprises values representing the expression level of one or more genes whose expression is associated with the addictive disorder in subjects affected by the addictive disorder, providing a test expression profile comprising values representing the expression level of said one or more genes in a sample comprising cells that been contacted with the compound; and determining whether said test expression profile shares sufficient similarity to said reference profile.
  • the test expression profile is sufficiently similar to the reference profile, it is predicted that the compound will be addictive in humans.
  • the invention identifies genes that are differentially expressed in cocaine overdose victims in comparison with control subjects, and provides a method of screening for therapeutic compounds for use in treating addictive disorders using these genes.
  • the method comprises screening for modulators of genes that are differentially expressed in cocaine overdose victims in comparison with control subjects.
  • Reports have shown that common molecular pathways, including the stimulation of dopamine transmission in the limbic system, underlie the addictive nature of a number of drugs including opiates, ethanol, nicotine, amphetamine, and cocaine. See, for example, Koob and Nester (1997) Journal ofNeuropsychiatry and Clinical Neurosciences 9:482-497; Wise and Bozarth (1987) Psychological Review 94:469-492; Di Chiara and Imperato (1988) Proc. Natl.
  • genes having differential expression in cocaine overdose victims are targets for the diagnosis and treatment of a number of addictive disorders.
  • the invention provides methods for evaluating a candidate drug to determine whether it will have therapeutic efficacy in treating an addictive disorder.
  • the method comprises the steps of determining the expression level of one or more genes in a first sample from a subject affected by an addictive disorder prior to treatment with the candidate drug, wherein expression of said one or more genes is associated with the addictive disorder; determining the expression level of said one or more genes in a second sample from the subject following treatment with the candidate drug, and comparing the expression level measured before and after the treatment to determine the change in the expression level of said genes following treatment with the candidate drug. If the drug results in a change in the expression levels of the genes associated with the addictive disorder, it results in a prediction that the candidate drug will have therapeutic efficacy in treating the disorder.
  • compositions for use in the methods of the invention are also provided.
  • Compositions of the invention include an array comprising a substrate having a plurality of addresses, where each address has disposed thereon a capture probe that can specifically bind a nucleic acid molecule that is differentially expressed in subjects affected by cocaine addiction and a computer-readable medium having a digitally-encoded expression profile comprising one or more values representing the expression levels of a gene that is differentially expressed in cocaine overdose victims.
  • compositions of the invention also include a kit for predicting whether a compound will be addictive in humans and a kit for evaluating the therapeutic efficacy of a candidate drug.
  • the kits comprise an array having multiple addresses, wherein each address has a capture probe that can specifically bind a nucleic acid molecule that is differentially expressed in subjects affected by an addictive disorder; and a computer-readable medium having a digitally-encoded expression profiles having values representing the expression of a nucleic acid molecules detected by the array.
  • the methods of the invention have been applied to cocaine addiction to identify candidate targets for improving currently available treaments. Accordingly, one aspect of the invention is the use of the identified target genes in drug screening for drugs to treat addictive disorders.
  • FIGURES Figures 1A and IB A. Ionotropic glutamate receptor subunit protein levels in VTA and 1-SN of cocaine overdose victims. Membrane fractions were isolated as described in the Experimental Procedures and 10 mg were separated on 10% SDS- PAGE to assess glutamate receptor subunit immunoreactivity. Data are expressed as mean ( ⁇ S.E.M.) of the percent of control values per amount of protein loaded. Asterisks indicate a significant difference (PO.05).
  • B Representative bands from two cocaine overdose victims (+) and two control subjects (-) for each subunit.
  • Figures 2 A and 2B A. CREB protein levels in the cytosolic and nuclear fragments of VTA and 1-SN in cocaine overdose victims. Cytoplasmic and nuclear fractions were isolated as described in the Experimental Procedures and 15 mg were separated on 10% SDS-PAGE to assess CREB immunoreactivity. Phospho-CREB immunoreactivity was not detectable in either the cytoplasmic or nuclear fractions. Data are expressed as mean ( ⁇ S.E.M.) of the percent of control values. * indicate significant difference (P ⁇ 0.05). B. Representative bands from two cocaine overdose victims (+) and two control subjects (-).
  • FIGS 3 A and 3B A. Levels of G-protein subunits in VTA and 1-SN of cocaine overdose victims. Cytoplasmic fractions were isolated as described in the Methods and 10 mg were separated on 10% SDS-PAGE to assess G-protein subunit immunoreactivity. Data are expressed as mean ( ⁇ S .E.M.) of the percent of control values per amount of protein loaded. Asterisks indicate a significant difference (P ⁇ 0.05).
  • B Representative bands from two cocaine overdose victims (+) and two control subjects (-).
  • the present invention demonstrates changes in gene expression levels in neuronal populations in subjects affected by an addictive disorder in comparison with control subjects. Changes in gene expression levels were detected in human postmortem samples, providing direct evidence of addiction-associated changes in gene expression in human subjects. Changes in gene experssion levels were also detected follwing binge cocaine self administration in rats.
  • the identification of genes that are differentially expressed in subjects affected by addictive disorders provides methods for identifying genes and their expression products as targets in the diagnosis and treatment of addictive disorders. According to the invention, genes whose expression level is significantly increased or significantly decreased in subjects affected by an addictive disorder in comparison with control subjects are identified, along with their expression products, as screening targets for use in methods of identifying drugs for treating addictive disorders.
  • the methods of identifying genes whose expression is correlated with addiction provide an improvement in a method for screening for therapeutic compounds for use in treating addictive disorder.
  • the improvement comprises the use of a target identified by the method described above.
  • the identification of genes whose expression is correlated with addiction is also in creating expression profiles correlated with addiction.
  • the present invention provides methods for producing an expression profile having values representing the expression levels of genes whose expression is correlated with addictive disorders.
  • the method comprises determining the expression level of one or more genes in a sample from subjects affected by an addictive disorder, determining the expression levels of the same genes in control subjects, and identifying genes whose expression level is significantly increased or significantly decreased in subjects affected by the addictive disorder in comparison with control subjects.
  • the invention also encompasses methods for predicting whether a compound will be addictive in humans.
  • the methods comprise the steps of providing a reference expression profile associated with an addictive disorder, where the expression profile comprises values representing the expression level of one or more genes whose expression is associated with the addictive disorder in subjects affected by the addictive disorder, providing a test expression profile comprising values representing the expression level of said one or more genes in a sample comprising cells that been contacted with the compound; and determining whether said test expression profile shares sufficient similarity to said reference profile.
  • the test expression profile is sufficiently similar to the reference profile, it is predicted that the compound will be addictive in humans.
  • the invention identifies genes that are differentially expressed in cocaine overdose victims in comparison with control subjects, and provides a method of screening for therapeutic compounds for use in treating addictive disorders using these genes.
  • the method comprises screening for modulators of genes that are differentially expressed in cocaine overdose victims in comparison with control subjects.
  • the invention provides methods for evaluating a candidate drug to determine whether it will have therapeutic efficacy in treating an addictive disorder.
  • the method comprises the steps of determining the expression level of one or more genes in a first sample from a subject affected by an addictive disorder prior to treatment with the candidate drag, wherein expression of said one or more genes is associated with the addictive disorder; determining the expression level of said one or more genes in a second sample from the subject following treatment with the candidate drag, and comparing the expression level measured before and after the treatment to determine the change in the expression level of said genes following treatment with the candidate drug. If the drug results in a change in the expression levels of the genes associated with the addictive disorder, it results in a prediction that the candidate drug will have therapeutic efficacy in treating the disorder.
  • the present invention provides methods for predicating the therapeutic efficacy and the potential for addiction of a candidate drug.
  • therapeutic efficacy it is intended the ability of the drag to alleviate (e.g., mitigate, decrease, reduce) at least one of the symptom associated with the condition to be treated.
  • an “addictive” compound it is intended a compound that creates a certain degree of euphoria in the subject and has a strong potential for addiction.
  • compositions for use in the methods of the invention are also provided.
  • Compositions of the invention include an array comprising a substrate having a plurality of addresses, where each address has disposed thereon a capture probe that can specifically bind a nucleic acid molecule that is differentially expressed in subjects affected by cocaine addiction and a computer-readable medium having a digitally-encoded expression profile comprising one or more values representing the expression levels of a gene that is differentially expressed in cocaine overdose victims.
  • compositions of the invention also include a kit for predicting whether a compound will be addictive in humans and a kit for evaluating the therapeutic efficacy of a candidate drug.
  • the kits comprise an array having multiple addresses, wherein each address has a capture probe that can specifically bind a nucleic acid molecule that is differentially expressed in subjects affected by an addictive disorder; and a computer-readable medium having a digitally-encoded expression profiles having values representing the expression of a nucleic acid molecules detected by the array.
  • an "expression level” or “level of expression” is a value that corresponds to a measurement of the relative or absolute abundance of a gene expression product. Such values may include measurements of RNA levels or protein abundance. Thus, an expression level can be a value that reflects the transcriptional state or the translation state of a gene.
  • the transcriptional state of a sample includes the identities and abundance of the RNA species, especially mRNAs present in the sample.
  • the transcriptional state can be conveniently determined by measuring transcript abundance by any of several existing gene expression technologies.
  • Translational state includes the identities and abundance of the constituent protein species in the sample.
  • an "expression profile" comprises one or more values corresponding to a measurement of the relative or absolute abundance of a gene expression product. Such values may include measurements of RNA levels or protein abundance. Thus, the expression profile can comprise values representing the measurement of the transcriptional state or the translational state of the gene. See, U.S. Pat. Nos. 6,040,138, 5,800,992, 6,020135, 6,344,316, and 6,033,860, which are hereby incorporated by reference in their entireties.
  • the transcriptional state of a sample includes the identities and relative abundance of the RNA species, especially mRNAs present in the sample.
  • RNA species in the sample are measured, but at least a sufficient fraction to characterize the transcriptional state of the sample is measured.
  • the transcriptional state can be conveniently determined by measuring transcript abundance by any of several existing gene expression technologies.
  • Translational state includes the identities and relative abundance of the constituent protein species in the sample.
  • the expression profiles of the present invention are generated from samples from subjects affected by an addictive disorder.
  • addictive disorder is a disorder involving a pattern of compulsive drag use characterized by a continued craving for a drug and the need to use the drug for effects other than pain relief.
  • addictive disorders of the present invention include, but are not limited to, addiction to cocaine, addiction to opiates (e.g., heroin, methadone, morphine, oxycodone), addiction to stimulants
  • samples from the subject affected by an addictive disorder used to generate the expression profiles of the present invention can be derived from a variety of sources including, but not limited to, single cells, a collection of cells, tissue, or cell culture.
  • the tissue or cell source may include a post-mortem sample, cell culture, or a single cell.
  • Samples may comprise at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% cells having differential expression in addictive disorders, with a preference for samples having a higher percentage of such cells.
  • these cells are neuronal cells.
  • these cells are ventral tegmental area cells or lateral substantia nigra cells.
  • the sample is of human origin, while in other embodiments it is derived from a non- human model system for addictive disorders.
  • model systems include rodent model (e.g. self- admimstration models, conditioned place preference models, drag discrimination models, intracranial self-stimulation models, or locomoter activity models) and non-human primate models (e.g., rhesus monkey, squirrel monkey, baboon) for self-administration or drug discrimination.
  • rodent model e.g. self- admimstration models, conditioned place preference models, drag discrimination models, intracranial self-stimulation models, or locomoter activity models
  • non-human primate models e.g., rhesus monkey, squirrel monkey, baboon
  • model systems are described, for example, in Hemby et al. (1997) Neuropharmcological basis of drag reinforcement, in Drug Addiction and its Treatment: Nexus of
  • the present invention also provides for measuring the level of gene in expression in control subjects.
  • the control subjects of the invention are subjects that are not affected by drug addiction.
  • the control subjects matched to the test subjects affected by the addictive disorder by ensuring that both the control subject and the test subject are of a similar age.
  • other characteristics may also be matched between the control and test subjects such as race and sex. Matching characteristics of the test and control subjects reduces the likelihood that gene expression changes will results from a factor other than a difference in the use of addictive substances.
  • the expression profiles comprise values representing the expression levels of genes that are differentially expressed in subjects affected by an addictive disorder in comparison with control subjects.
  • the term "differentially expressed” as used herein means that the measurement of a cellular constituent varies in two or more samples.
  • the cellular constituent may be upregulated in a sample from a subject having one physiologic condition in comparison with a sample from a subject having a different physiologic condition, or down regulated in a sample from a subject having one physiologic condition in comparison with a sample from a subject having a different physiologic condition.
  • the expression level of the gene may be significantly increased or significantly decreased in subjects affected by an addictive disorder in comparison with control subjects.
  • a "significant” change in expression level it is intended a change in expression level that is statistically significant.
  • a statistical test may be used to test whether a change in expression level measured for a gene after treatment is more likely to result from an actual change in the expression of the gene rather than from any variability present in the experimental system.
  • the present invention provides groups of genes that are differentially expressed in cocaine overdose victims in comparison with age-matched control subjects. These genes were identified based on gene expression levels for 8700 probes in 8 post-mortem tissue samples from cocaine overdose victims and 8 post- mortem tissue samples from control subjects, and on mRNA and protein levels for candidate genes (Table 2). Values representing the expression levels of the nucleic acid molecules detected were analyzed to identify those that were significantly up- or down-regulated in cocaine overdose victims in comparison with control subjects. See the Experimental section. The present provides genes with a differential level of expression in cocaine overdose victims compared to control subjects. These differentially expressed genes are selected from the genes shown in Tables 3, 4, and 5. These genes and their expression products are useful as markers to detect the presence of an addictive disorder in a patient.
  • the present also provides glutamate receptor subunits with a differential level of expression in rats following binge access to cocaine in comparison with controls.
  • the genes encoding these differentially-expressed receptor subunits are shown in Table 6. These genes and their expression products are useful as markers to detect the presence of an addictive disorder in a patient, and as screening targets for compounds to treat addicitive disorders.
  • Each expression profile of the invention contains a sufficient number of values such that the profile can be used to distinguish samples correlated with addictive disorders from control samples.
  • the expression profiles comprise only one value.
  • the expression profile comprises more than one value corresponding to a differentially expressed gene, for example at least 2 values, at least 3 values, at least 4 values, at least 5 values, at least 6 values, at least 7 values, at least 8 values, at least 9 values, at least 10 values, at least 11 values, at least 12 values, at least 13 values, at least 14 values, at least 15 values, at least 16 values, at least 17 values, at least 18 values, at least 19 values, at least 20 values, at least 22 values, at least 25 values, at least 27 values, at least 30 values, at least 35 values , at least 40 values, at least 45 values, at least 50 values, at least 75 values, at least 100 values, at least 125 values, at least 150 values, at least 175 values, at least 200 values, at least 250 values, at least 300 values, at least 400 values, at least
  • the diagnostic accuracy of the methods of the present invention will vary based on the strength of the correlation between the expression levels of the differentially expressed genes and the associated physiologic condition.
  • the values in the expression profiles represent the expression levels of genes whose expression is strongly correlated with the physiologic condition, it may be possible to use fewer number of values in the expression profile and still obtain an acceptable level of diagnostic or prognostic accuracy.
  • a gene whose expression level is "correlated with" a particular physiologic condition it is intended a gene whose expression shows a statistically significant correlation with the physiologic condition.
  • the significance of the correlation between the expression level of a differentially expressed gene and a particular physiologic state may be determined by a statistical test of significance. Such methods are known in the art and examples are provided elsewhere herein. Methods for detemiining the strength of a correlation between the expression level of a differentially-expressed gene and a particular physiologic state are also reviewed in Holloway et al. (2002) Nature Genetics Suppl. 32:481-89, Churchill (2002) Nature Genetics Suppl. 32:490-95, Quackenbush (2002 ⁇ Nature Genetics Suppl. 32: 496-501 ; Slonim (2002) Nature Genetics Suppl.
  • Each value in the expression profiles of the invention is a measurement representing the absolute or the relative expression level of a differentially expressed genes.
  • the expression levels of these genes may be determined by any method l ⁇ iown in the art for assessing the expression level of an RNA or protein molecule in a sample.
  • RNA expression levels of RNA may be momtored using a membrane blot (such as used in hybridization analysis such as Northern, Southern, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids). See U.S. Patent Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which are expressly incorporated herein by reference.
  • the gene expression monitoring system may also comprise nucleic acid probes in solution.
  • microarrays are used to measure the values to be included in the expression profiles. Microarrays are particularly well suited for this purpose because of the reproducibility between different experiments.
  • DNA microarrays provide one method for the simultaneous measurement of the expression levels of large numbers of genes. Each array consists of a reproducible pattern of capture probes attached to a solid support. Labeled RNA or DNA is hybridized to complementary probes on the array and then detected by laser scanning. Hybridization intensities for each probe on the array are determined and converted to a quantitative value representing relative gene expression levels. See, the Experimental section. See also, U.S. Pat. Nos.
  • High-density oligonucleotide arrays are particularly useful for determining the gene expression profile for a large number of RNA's in a sample.
  • total mRNA isolated from the sample is converted to labeled cRNA and then hybridized to an oligonucleotide array. Each sample is hybridized to a separate array. Relative transcript levels are calculated by reference to appropriate controls present on the array and in the sample. See, for example, the Experimental section.
  • the values in the expression profile are obtained by measuring the abundance of the protein products of the differentially-expressed genes.
  • the abundance of these protein products can be dete ⁇ nined, for example, using antibodies specific for the protein products of the differentially-expressed genes.
  • antibody refers to an immunoglobulin molecule or immunologically active portion thereof, i.e., an antigen-binding portion.
  • immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
  • the antibody can be a polyclonal, monoclonal, recombinant, e.g., a chimeric or humanized, fully human, non-human, e.g., murine, or single chain antibody. In a preferred embodiment it has effector function and can fix complement.
  • the antibody can be coupled to a toxin or imaging agent.
  • a full-length protein product from a differentially-expressed gene, or an antigenic peptide fragment of the protein product can be used as an immunogen.
  • Preferred epitopes encompassed by the antigenic peptide are regions of the protein product of the differentially expressed gene that are located on the surface of the protein, e.g., hydrophilic regions, as well as regions with high antigenicity.
  • the antibody can be used to detect the protein product of the differentially expressed gene in order to evaluate the abundance and pattern of expression of the protein. These antibodies can also be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given drug.
  • Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance (i.e., antibody labeling).
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, biolummescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, b-galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • examples of biolummescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 L 131 I, 35 S or 3 H.
  • the subject profile is compared to the reference profile to determine whether the subject expression profile is sufficiently similar to the reference profile. Any method known in the art for comparing two or more data sets to detect similarity between them may be used to compare the subject expression profile to the reference expression profiles.
  • Any method known in the art for comparing two or more data sets to detect similarity between them may be used to compare the subject expression profile to the reference expression profiles.
  • statistical tests may be performed to determine whether the similarity between the subject expression profile and the reference expression profile is likely to have been achieved by a random event. Examples of such a statistical tests are described in the Experimental section; however, any statistical test that can calculate the likelihood that the similarity between the subject expression profile and the reference profile results from a random event can be used.
  • the accuracy of assigning a subject to a risk group based on similarity between an expression profile for the subject and an expression profile for the risk group depends in part on the degree of similarity between the two profiles. Therefore, when more accurate diagnoses are required, the stringency with which the similarity between the subject expression profile and the reference profile is evaluated should be increased.
  • the p-value obtained when comparing the subject expression profile to a reference profile that shares sufficient similarity with the subject expression profile is less than 0.20, less than 0.15, less than 0.10, less than 0.09, less than 0.08, less than 0.07, less than 0.06, less than 0.05, less than 0.04, less than 0.03, less than 0.02, or less than 0.01.
  • compositions that are useful in the disclosed methods of identifying screening targets. These compositions include arrays comprising a substrate having a capture probes that can bind specifically to nucleic acid molecules that are differentially expressed in subjects affected by addictive disorders in comparison with control subjects. Also provided is a computer-readable medium having digitally encoded reference profiles useful in the methods of the claimed invention. The invention also encompasses kits comprising an array of the invention and a computer-readable medium having digitally-encoded reference profiles with values representing the expression of nucleic acid molecules detected by the arrays. These kits are useful for methods of evaluating the therapeutic efficacy of drags for treating addictive disorders, and methods for predicting whether a compound will be addictive in humans.
  • arrays comprising capture probes for detecting the differentially expressed genes of the invention.
  • array is intended a solid support or substrate with peptide or nucleic acid probes attached to said support or substrate.
  • Arrays typically comprise a plurality of different nucleic acid or peptide capture probes that are coupled to a surface of a substrate in different, known locations.
  • arrays may generally be produced using mechanical synthesis methods or light directed synthesis methods, which incorporate a combination of photolithographic methods and solid phase synthesis methods.
  • arrays may be peptides or nucleic acids on beads, gels, polymeric surfaces, fibers such as fiber optics, glass or any other appropriate substrate, see U.S. Pat. Nos. 5,770,358, 5,789,162, 5,708,153, 6,040,193 and 5,800,992, each of which is hereby incorporated in its entirety for all purposes.
  • Arrays may be packaged in such a manner as to allow for diagnostics or other manipulation of an all-inclusive device. See, for example, U.S. Pat. Nos. 5,856,174 and 5,922,591 herein incorporated by reference.
  • the arrays provided by the present invention comprise capture probes that can specifically bind a nucleic acid molecule that is differentially expressed in cocaine overdose victims.
  • each capture probe in the array detects a nucleic acid molecule selected from the nucleic acid molecules designated in Tables 3, 4, and 5.
  • the designated nucleic acid molecules include those differentially expressed in subjects affected by addictive disorders in comparison with control subjects.
  • the arrays of the invention comprise a substrate have a plurality of addresses, where each addresses has a capture probe that can specifically bind a target nucleic acid molecule.
  • the number of addresses on the substrate varies with the purpose for which the array is intended.
  • the arrays may be low-density arrays or high-density arrays and may contain 4 or more, 8 or more, 12 or more, 16 or more, 20 or more, 24 or more, 32 or more, 48 or more, 64 or more, 72 or more 80 or more, 96, or more addresses, or 192 or more, 288 or more, 384 or more, 768 or more, 1536 or more, 3072 or more, 6144 or more, 9216 or more, 12288 or more, 15360 or more, or 18432 or more addresses.
  • the substrate has no more than 12, 24, 48, 96, or 192, or 384 addresses, no more than 500, 600, 700, 800, or 900 addresses, or no more than 1000, 1200, 1600, 2400, or 3600 addressees.
  • the invention also provides a computer-readable medium comprising one or more digitally-encoded expression profiles, where each profile has one or more values representing the expression of a gene that is differentially expressed in a subject affected by an addictive disorder.
  • the digitally-encoded expression profiles are comprised in a database. See, for example, U.S. Patent No. 6,308,170.
  • the database may be screened to identify genes to be used as targets in a screen for compounds useful for treating addictive disorders.
  • kits useful for evaluating the therapeutic efficacy or potential for addition of compounds or candidate drugs comprise an array and a computer readable medium.
  • the array comprises a substrate having addresses, where each address has a capture probe that can specifically bind a nucleic acid molecule that is differentially expressed in subjects affected by an addictive disorder in comparison with control subjects.
  • the results are converted into a computer-readable medium that has digitally-encoded expression profiles containing values representing the expression level of a nucleic acid molecule detected by the array.
  • the methods and compositions of the invention may be used to screen test compounds to identify therapeutic compounds useful for the treatment of addictive disorders.
  • the test compounds are screened in a sample comprising primary cells or a cell line representative of a neuronal cell type.
  • the expression levels in the sample of one or more of the differentially-expressed genes of the invention are measured using methods described elsewhere herein. Values representing the expression levels of the differentially-expressed genes are used to generate a test expression profile.
  • This subject expression profile is then compared to a reference profile associated with the addictive disorder represented by the sample to determine the similarity between the subject expression profile and the reference expression profile. Differences between the subject expression profile and the reference expression profile may be used to determine whether the test compound has therapeutic efficacy in treating an addictive disorder.
  • test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one- compound' library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145). Examples of methods for the synthesis of molecular libraries can be found in the art, for example in DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90:6909; Erb et al.
  • Candidate compounds include, for example, 1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam et al. (1991) Nature 354:82-84; Houghten et al. (1991) Nature 354:84- 86) and combinatorial chemistry-derived molecular libraries made of D- and/or L- configuration amino acids; 2) phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang et al.
  • peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam et al. (1991) Nature 354:82-84; Houghten et al. (1991) Nature 354:84- 86) and combinatorial chemistry-derived molecular libraries made of
  • the present invention discloses a number of genes that are differentially expressed in cocaine overdose victims in comparison with control subjects. These differentially-expressed genes are shown in Tables 3, 4, and 5. Because the expression of these genes is associated with drag addiction, these genes may play a role in the development of addiction. Accordingly, these genes and their gene products are potential therapeutic targets that are useful in methods of screening test compounds to identify therapeutic compounds for the treatment of addictive disorders.
  • the differentially-expressed genes of the invention may be used in cell-based screening assays involving recombinant host cells expressing the differentially- expressed gene product.
  • the recombinant host cells are then screened to identify compounds that can activate the product of the differentially-expressed gene (i.e. agonists) or inactivate the product of the differentially-expressed gene (i.e. antagonists).
  • Array analysis revealed significant up- regulation of numerous transcripts in the VTA, but not 1-SN, of cocaine overdose victims including NMDARI , GluR2, GluR5 and KA2 receptor mRNA (PO.05). No significant alterations between overdose victims and controls were observed for GluRl, R3 or R4 mRNA levels.
  • Western blot analysis revealed VTA-selective up- regulation of CREB (PO.01) NMDARI (PO.01), GluR2 (PO.05), GluR5 (PO.01), and KA2 (PO.05) protein levels of cocaine overdose victims.
  • iGluR ionotropic glutamate receptor
  • tissue punches were dissected from the VTA and 1-SN of the contralateral hemisphere used for RNA analysis in eight cocaine overdose victims (34.4 ⁇ 2.7 yr; 5WM, 3BM; PMI: 12.5 ⁇ 1.1 hr) and seven age-matched, drug free controls (37 ⁇ 2.7 yr; 6 WM, 1 WF; PMI: 12.6 ⁇ 0.7 hr), representing a subset of subjects from whom tissue was used in the RNA experiments.
  • COC cocaine
  • CE cocethylene
  • COD cocaine overdose
  • CTR control
  • BE benzylecognine
  • ND not detectable
  • NA not available
  • PMI post-mortem interval
  • LC lidocaine
  • EtOH ethanol
  • DHPA diphenhydramine
  • Brain tissue was cryopreserved using a procedure described in Hardy et al. 1983 J Neurochem. 40: 608-614; and Dodd et /. 1 Neurochemical Pathology 4, 177-198, both of which are herein incorporated by reference.
  • One hundred mg punches were dissected from the blocks containing the VTA (posterior region of slab) and SN (anterior region containing the pars lateralis and the pars medialis) - one hemisphere for RNA and the contralateral hemisphere for protein analysis.
  • Possible neuronal loss, ischemic cell changes, and reactive gliosis were assessed using semiquantitative ratings by the neuropathologists and found to be negligible in all cases used in the present study.
  • RNA isolation and amplification Micro-FastTrack® brand RNA isolation kits (Invitrogen; Carlsbad, Cah " fornia) were used to isolate polyadenylated RNA from the VTA and 1-SN. mRNA yields ranged from 1 to 2 mg/100 ng of frozen tissue. Due to the amount of mRNA, samples from each subject were amplified using a modification of the aRNA amplification procedure described previously in Ginsberg et al. (2000) Ann Neurol. 48:77-87; Hemby et al. (2002) Archives of General Psychiatry 59:631-640; and Hemby et al.
  • Oligo(dT)-T7- primer/promoter 500 ng
  • [AAACGACGGCCAGTGAATTGTAAT ACGACTCACTATAGGCGC(T)24] (SEQ ID NO:l) was hybridized to poly(A+)mRNA in the presence of 500 mM each of dNTPS and RNase free water for 30 minutes at 65°C, then quick cooled on ice for 5 minutes.
  • 10 mM DTT, IX first strand buffer and 20U RNAsin were added and samples were incubated at 42°C for 2 minutes as described in Van Gelder et al. (1990) Proc.
  • RNA antisense RNA
  • aRNA was synthesized using T7 RNA polymerase (EpiCentre Technologies; Madison, WI) in IX transcription buffer (Epicentre Technologies), 10 mM DTT, 250 mM each of NTPs and 20U RNAsin at 37°C overnight.
  • IX transcription buffer Epicentre Technologies
  • aRNA was reverse transcribed using Superscript II in the presence of IX 1st strand buffer, 250 mM each of dNTPs, 100 ng of random hexamers and 20U RNAsin incubated at 37°C for one hr.
  • samples were incubated with 10U Klenow fragment, 250 mM each of dNTPs, 500 ng of the aforementioned oligo dT-T7 primer-promoter, and 5U T4 DNA polymerase at 16°C overnight. Samples were phenol/chloroform extracted, ethanol precipitated and drop dialyzed for 15 min.
  • reaction conditions were identical for the first round except for the inclusion of 30 mCi [a- 33 P]-UTP (2500 Ci/mmol; Amersham Pharmacia Biotech; Piscataway, NJ), 10 mM of UTP and 250 mM each of CTP, GTP and ATP.
  • the aRNA procedure is a linear amplification process with minimal change in the relative abundance of the mRNA population in the native state of the neuron. mRNA can be reliably amplified from small amounts of fixed tissue including individual neurons and neuronal processes (Van Gelder et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87:1663-1667; Eberwine et al.
  • Reverse Northern Blots were prepared on nylon membranes containing candidate genes including dopamine receptors (e.g. Dl, D2, D4, D5 and DAT), G- protein subunits (ail, ai2, as, az, aq, ao, ⁇ , ⁇ l and ⁇ 2), glutamate receptor mRNAs (mGluR3, GRIA1-4, GRD 5, 7 and GRIN1), GABA A receptor subunits ( ⁇ l, ⁇ 2, ⁇ l, ⁇ 3, ⁇ 2, ⁇ , ⁇ , and ⁇ ), regulators of G-protein signaling (RGS 1-7, 9, 10, 12, 13, 16) and other transcripts (cannabinoid 1 receptor, cocame-amphetamine regulated transcript (CART), serotonin 2 and serotonin 3 receptors, and tyrosine hydroxylase).
  • dopamine receptors e.g. Dl, D2, D4, D5 and DAT
  • G- protein subunits as, az, aq,
  • Inserts were amplified in 96 well plates using PCR with GF200 primers under the following conditions: 95°C for 5min (1 cycle); 95°C for 30 sec, 52°C for 45 sec, and 72°C for 2 min (40 cycles); and 72°C for 7 min (1 cycle).
  • PCR samples were purified (MultiscreenTM PCR filter plate, Millipore, Bedford, MA) and aliquots were electrophoresed on a 1% agarose gel (IX TAE, 0.05% ethidium bromide) at 5V/cm for PCR band size verification. Gel images were captured by digital camera and archived.
  • PCR product concentration was determined by spectrofluorometry (Gemini Microplate Spectrofluorometer, Molecular Devices, Sunnyvale, CA) using a 1:5000 dilution of SYBR 1 Green/TE and an aliquot of the PCR product. Values were compared to concentrations of known DNA standards for quantitation. 250 ng of each amplified insert was spotted on Nytran SuPerCharge ® nylon transfer membrane (Schleicher and Schuell, Keene, New Hampshire) using a 96 well dot blot apparatus (Schleicher and Schuell; Minifold I). DNA was crosslinked to the membrane by ultraviolet radiation.
  • Arrays were pre-hybridized with ULTRAhybTM solution (Ambion; Austin, TX) in hybridization bottles for 1 hr at 42°C.
  • P-labelled aRNA probes from each subject were heat denatured for 5 min at 65°C, and then immediately added to their respective bottles and allowed to hybridize for 18-24 hrs at 42°C in a rotisserie hybridization oven.
  • membranes were washed twice with 2X SSC/0.1% SDS and 0.1X SSC/0.1% SDS for 20 min each at 42°C. Labeled hybridized products were detected using phosphorimager screens, and hybridization signal intensities were analyzed using ImageQuantTM software (Amersham Biosciences; Sunnyvale, CA).
  • Tissue samples were dounce homogenized in 10 mM HEPES, 10 mM NaCl, 1 mM KH2PO4, 5 mM NaHCO 3 , ImM CaCl 2 , 0.5 mM MgCl 2 , 5 mM EDTA and the following protease inhibitors (PI): ImMPMSF, lO mMbenzan ⁇ dine, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 1 mg/ml pepstatin and centrifuged at 7500 RPM for 5 min.
  • PI protease inhibitors
  • Protein bands were visualized on a Kodak XAR-5 film with enhanced chemiluminescence (ECL PlusTM, Amersham Pharmacia Biotech).
  • Primary antibodies were as follows: mouse monoclonal antibodies directed against NMDARI (Chemicon International, Temecula, CA) and GluR5 (Upstate Biotechnologies, Waltham, MA); rabbit polyclonal antibodies directed against GluRl, GluR2/3, KA2, G s, Gail/2, G ⁇ , CREB , phospho-CREB (Upstate Biotechnologies) and FRA-2 (L- 15 ; Santa Cruz
  • Specific signal (minus background) of probe bound to each PCR product was expressed as a ratio of the total hybridization intensity of spots of the custom-designed array, thereby minimizing variations due to differences in the specific activity of the probe and the absolute quantity of probe present (Ginsberg et al. (2000) Ann Neurol. 48:77-87; Hemby et al. (2002) Archives of General Psychiatry 59:631-640; and Hemby et al. (2003) Journal of Comparative Neurology 456: 176-183).
  • Array data were analyzed using a two way ANOVA with group and anato ical region as the fixed effects and normalized hybridization intensity as the dependent measure. Post hoc analyses were conducted as needed using Tukey's Test and the null hypothesis was rejected when P ⁇ 0.05.
  • Western blot data were analyzed using t-tests comparing the band intensities between cocaine overdose victims and controls for each respective protein. Data were graphically depicted as percent of the controls for each respective protein. Null hypotheses were rejected when P
  • Custom-designed macroarrays containing 81 cDNAs were used to evaluate gene expression changes in the VTA and 1-SN of cocaine overdose victims and controls.
  • RNA processing Fas-related antigen 2 (FRA2); early growth response (EGR) 2 and 3; protein kinase interferon-inducible double stranded RNA dependent (PRKR); CUG triplet repeat RNA binding protein 2 (CUGBP2); RNA polymerase JJ-DNA directed- polypeptide C 33kDa (POLR2C); suppressor of RNA polymerase B7 (RNApH); TATA box binding protein- associated factor, RNA polymerase IT, H, 30kD (TBP-af30)) did not reveal any significant difference between cocaine overdose victims and controls in either the VTA or 1-SN.
  • Protein expression Western blot analysis was performed on protein homogenates from individual subjects to assess the correlation of changes in mRNA levels with protein levels. From the gene expression analysis, proteins for NRl, GluRl, GluR2, GluR5, KA2, Gail, Gas, and G ⁇ were selected based on the availability of selective antibodies, hi addition, protein levels of cAMP responsive element binding protein (CREB), phosphorylated CREB, and FRA2 were evaluated based on their regulation in animal models of drug abuse (Carlezon et al. (1998) Science 282:2272-2275; Self et al. (1998) Journal of Neuroscience 18:1848-1859; Pliakas et al. (2001) Journal ofNeuroscience 21:7397- 7403). h membrane fractions, NRl immunoreactive protein was increased 111.2% (PO.05), GluR2/3 by 69.1% (PO.05), GluR5 by 507.9% (P .01), and KA2 by 72.1%
  • the present invention demonstrates that increased NRl protein levels in cocaine overdose victims may be induced by acute or sub-acute cocaine exposure or possibly reflect the last acute cocaine binge prior to death. Upregulation of mRNA and protein levels of GluR2, GluR5 and KA2 are indicative of chronic cocaine use and/or cocaine overdose in humans.
  • GluRl -4 and NRl immunoreactivity was found to be localized almost exclusively on dopaminergic neurons (Paquet et al. 1997 J Neurosci. 17:1377-1396); however, less is known about the localization of kainate subunits in these regions.
  • Glutamatergic afferents in the midbrain originate from the medial frontal cortex, subthalamic and pedunculopontine tegmental nuclei (Christie et al. (1985) Brain Research 333:169-172.; Sesack et al.
  • GluR2 subunit protein levels were increased in the hippocampus of alcoholics (Breese et al. (1995) Brain Research 674:82-90) and GluR5 mRNA and protein levels were increased in the dorsal prefrontal cortex of cocaine treated rats (Toda et al. (2002) Journal of Neuroehemistry 82, 1290-1299).
  • the upregulation observed in the present study may represent a molecular compensation for the effects of increased Ca2+ influx generated by the combined upregulation of NRl, GluR5 and KA2 (Paschen et al. (1994) Journal of Neuroehemistry. 63:1596-1602).
  • upregulation of the iGluR subunit mRNA and protein levels in the present study are likely attributable to changes in VTA dopaminergic neurons, suggest ng a neuroadaptive response to cocaine in these subjects.
  • Dopamine receptor mRNAs were not differentially regulated as a function of cocaine overdose, opening the possibility that intracellular signaling mechanisms may also contribute to the altered function of these neurons.
  • Neuroadaptations in the cAMP pathway have been implicated in mesolimbic brain regions as a function of cocaine exposure (Nestler et al. (1990) Journal of Neuroehemistry. 55:1079-1082; Terwilliger et al. (1991) Brain Research. 548:100-110; Striplin and Kalivas (1992) Brain Research. 579:181-186; Miserendino and Nestler (1995) Brain Research. 674:299-306; Carlezon et al. (1998) Brain Research. 674:299-306; Self et al.
  • G ⁇ mRNA levels may manifest as down-regulated protein levels in axonal targets of the mesoaccumbens dopamine neurons as shown previously in cocaine-treated rats (Wang et al. (1991) Journal of N eur oscience 17:5993-6000.
  • the present data contrast previous studies showing decreased ADP ribosylation and immunoreactivity G ⁇ i and G ⁇ o in the VTA of cocaine treated rats.
  • a significant increase in CREB mRNA and protem levels in the cytosolic fraction and a trend towards significance in the nuclear fraction in the VTA of cocaine overdose victims was observed - the first demonstration of increased CREB protein levels in the VTA of either humans or animal models as a function of cocaine exposure.
  • CREB activity may be regulated is by Ca2+ influx via NMDA receptors, demonstrating a requirement for CREB-NMDA receptor interactions in dopamine-regulated gene expression (Konradi et al. (1996) Journal ofNeuroscience 16, 4231-4239).
  • One of the transcripts regulated by CREB is CART (Dominguez et al. (2002)
  • RGS3 and RGS 12 mRNAs in the VTA of cocaine overdose victims represents another potential mechanism for cocaine-induced alterations in cell signaling.
  • Previous studies have shown elevated RGS 2, 3 and 5 mRNA levels in the striatum by acute amphetamine administration (Burchett et al. (1999) Journal of Neuroehemistry 72:1529-1533) and RGS 2 mRNA levels by cocaine in the hippocampus, cortex, and striatum (Ingi etal. (1998) Journal oj " Neuroscience 18:7178- 7188).
  • RGS proteins modulate/integrate G-protein and other intracellular signaling proteins, some of which reduce the duration of G-protein activation by increasing the rate of GTP hydrolysis.
  • RGS3 interacts with tlie G protein bg complex to accelerate the rate of recovery of N-type Ca2+ inhibition from Gbg thereby leading to increased Ca2+ conductance and potentially to increased excitability of VTA dopamine neurons.
  • Example 2 Microarray Analysis of Molecular Correlates of Cocaine Addiction
  • the present example describes the identification of genes that are differentially expressed in the VTA of cocaine overdose victims in comparison with age-matched controls.
  • mRNA from each subject was isolated as described above in Example 1. Equivalent concentrations of mRNA from each subject within the cocaine overdose group or the control group were combined. Pooled samples were reverse transcribed and hybridized to the microarrays, and the resulting image was analyzed to determine gene expression levels.
  • Table 4 Transcripts down-regulated in cocaine overdose victims.
  • Table 5 Transcripts up-regulated in cocaine overdose victims.
  • Functional clustering was used to assess differential gene expression in biochemical pathways (e.g., cAMP and phosphoinositol pathways), protein families (e.g. dopamine receptors); protein motifs (e.g., zinc finger, leucine zipper, etc.) and transcripts previously identified as associated with cocaine abuse in human and animal models.
  • biochemical pathways e.g., cAMP and phosphoinositol pathways
  • protein families e.g. dopamine receptors
  • protein motifs e.g., zinc finger, leucine zipper, etc.
  • Reverse Northern Analysis was used as the secondary screen for 192 transcripts that were identified as differentially expressed (> 1.8 fold) in the high density microarray or as candidate genes that did not appear on the microarray.
  • the resulting data are consistent with neuroadaptive response in intracellular signaling cascades associated with long-term cocaine-induced stimulation of dopamine receptors. Therefore, expression levels of a number of dopamine and signal transduction levels were determined by reverse Northern analysis to dete ⁇ riine the pattern of gene expression and indicate whether the VTA in cocaine overdose victims is preferentially affected as a consequence of long-term cocaine use.
  • mRNA from each subject for each group was hybridized separately to two reverse Northern blots each containing 96 candidate clones.
  • PCR was performed to amplify the inserts of the clones of interest. Approximately 250 ng of PCR material from each clone was spotted on Hybond XL nylon membrane. Probes were generated as described for Example 1 and hybridized to reverse Northern blots. Blots were exposed to phospho screens and desitometric analysis was performed using ImageQuant software.
  • cDNA from the clones are amplified, purified, and aliquoted into 96-well plates using a Quiagen BioRobot 3000. Universal primer pairs are used for PCR. Clones are amplified, and an aliquot is electrophoresed on an agarose gel for quality control, and any failed reaction (e.g. a reaction resulting in no or multiple bands) is repeated. cDNA is purified from the PCR reactions and an aliquot is used for fluorometric quantification. Samples are deleted in 50% DMS) to prevent evaporation during spotting. Plates containing the products are loaded onto a Cartesian Microarray and spotted at a concentration of 10 ng per spot.
  • Each clone is spotted three times in different areas of the array.
  • the arrayer uses a quill type spotter and is enclosed in a temperature and humidity controlled chamber. Pre-spotting 10 times increases the reproducibility of the amount of product spotted on arrays.
  • Microarrays are dried at 37°C for one hour, and are then irradiated by ultraviolet to cross-link the DNA. Microarrays are washed with 70% ethanol, dried at 37°C and stored in dessicators at 4°C until needed.
  • RNA is incubated at 70°C for 20 minutes, then quick cooled in ice for 5 minutes.
  • Oligo dT-24 400 ng
  • 10 mM DTT and reverse transcriptase buffer to a final concentration of IX are added to the mRNA.
  • the sample is incubated at 37°C for 30 minutes. On ⁇ l of
  • Hybridization of microarrays is performed using a Genomic Solutions GenTac Hybridization Staion. Both labeled probes are added to the hybridization chamber and are agitated genetly at 48°C for 24 hours. Following hybridization, a medium stringency was buffer (2X SSC/0.1% SDS) is applied to the slides with a buffer flow time of 45 seconds at 40°C for 6 minutes. A second was step with a buffer of higher stringency (0.5X SSC/0.1% SDS) is applied to the slides with a buffer flow time of 45 seconds at 40°C for 3 minutes. 2X SSC is used as the post wash buffer with a flow time of 40 seconds at 25° for 30 seconds each. Slides are dried at 25°C for 5 minutes before image analysis. 3. Image Analysis
  • microarrays are scanned using the GSI Lumonics ScanArray 5000.
  • Initial image analysis is performed using ScanArray software, and are subsequently analyzed using GLEAMS software (NuTec Services).
  • GLEAMS software NuTec Services
  • the levels of the Cy3 and Cy5 signals are determined, and the size and spacing of the dots on the microarray are defined and corrected for artifacts.
  • the background is calculated for each spot using the immediate area surrounding the target spot. Controls are used to adjust channel intensity and to calculate the overall expression mean of each gene.
  • Criteria for inclusion of spots for analysis include: 1) >4-fold signal intensity over background, 2) a ' symetric spot (not intensity) patter for each channel 3) lack of significant artifactual signal intensity (intensity recognition pattern algorithm in software and visual identification, and 4) hybridization present in 2 of the three spots per gene.
  • clones are amplified and analyzed on agarose gels as described above. 250 ng of each amplified insert is spotted on HyBond XL ® net nylon transfer membrane (Amersham Pharmacia Biotech) using a 96 well dot blot apparatus (Schleicher and Schuell). DNA is crosslinked to the membrane by ultraviolet radiation. This amount of cDNA ensures that the cDNA is in vast excess to the respective aRNA, so that the target will not limit hybridization.
  • Arrays will be hybridized for 48 hours at 44°C in a rotisserie hybridization oven with the following hybridization solution: 50% formamide, 5X SSC, 5X Denhard's solution, 0.1% SDS, 200 ng of sheared salmon sperm, and 1.0 mM sodim pyrophosphate. Following hybridization, slots blots are washed sequentially with 2X SSC/0.1% SDS, 0.5X SSC/0.1% SDS, and 0.1X SSC/0.1% SDS for 20 minutes each at 44°C. Labeled hybridized products are detected using phosphorimager cassettes, Image StormScanner and ImageQuant software (Molecular Dynamics).
  • the specific signal (corrected for background) of probe bound to each clone is expressed as a ratio of the total hybridization intensity of the array, thereby minimizing variations due to differences in the specific activity of the probe and the absolute quantity of probe present. Differential expression greater than 1.8-fold is accepted as above background and relevant for further examination. Relative changes in individual mRNAs for the respective brain regions is analyzed using a two-way ANOVA (analysis of variance between groups) with Group (cocaine overdose, controls) and Region (VTA vs. 1-SN; NAc vs. d-CP) as the fixed effects. The null hypothesis is rejected when P ⁇ 0.05. Post hoc analyses are conducted as needed using Fisher least significance difference.
  • Differentially expressed genes are grouped (clustered) into functional categories (e.g. signal transduction pathways, protein families such as receptors, RGS proteins, G-protein subunits, etc.) as well as to provide information on coordinate gene expression in a functional context.
  • functional categories e.g. signal transduction pathways, protein families such as receptors, RGS proteins, G-protein subunits, etc.
  • Differential gene expression is examined for relationships with indices of cocaine use (e.g. blood and brain concentrations; frequency and duration of use) by one-way ANOVA followed by post hoc analyses where appropriate.
  • mRNA abundance serves as the dependent measure.
  • Additional clinical data including age, postmortem interval, sex, and race are examined for effects on gene expressing using ANOVA or correlation analysis, as appropriate.
  • Ionotropic glutamate receptors are classified as NMD A (NRl, NR2A-D,
  • AMPA and kainate subunits contribute to fast neurotransmission
  • all three ionotropic subtypes are thought to play roles in long term potentiation.
  • subunit composition determines the functional properties of ionotropic glutamate receptors
  • alterations in ionotropic glutamate receptors in the VTA may indicate alterations in the excitability of dopamine transmission underlying long term biochemical and behavioral effects of cocaine which, in turn, may affect subsequent drug intake.
  • Rats were anesthetized with halothane and implanted with chronic indwelling venous catheters, as described previously (Hemby et al. (1999) J Pharmacol. Exp. Ther. 288:274-80; Hemby et al. (1995) J Pharmacol. Exp. Ther. 273:591-98; Hemby (1997b) Psychopharmacology ( e ⁇ ). 133:7-16).
  • Catheters were inserted into the right jugular vein, terminating just outside the right atrium and anchored to muscle near the point of entry into the vein. The distal end of the catheter was guided subcutaneously to exit above the scapulae through a Teflon shoulder harness.
  • the harness provided a point of attachment for a spring leash connected to a single channel swivel at the opposing end.
  • the catheter was threaded through the leash for attachment to the swivel where the fixed end of the swivel was connected to a syringe by polyethylene tubing.
  • Infusions were administered by a motor driven syringe pump controlled by a computer.
  • Infusions of methohexital 100 ⁇ l; 10 mg/kg; i.v.
  • Rats were randomly divided into two groups: (BINGE access and WITHDRAWAL) and allowed to self-administer cocaine (0.5 mg/infusion; 200 ⁇ l/infusion; 6.2 sec/infusion) during an 8 hr. self-administration sessions (dark phase of the light cycle) under a fixed ratio-5 (FR5); time out 20 seconds schedule of reinforcement.
  • a cocaine infusion was delivered and a 20-sec time-out was in effect.
  • Responding was initially maintained under an FR1 that was gradually increased to FR5. During the time-out, the lever light was extinguished, the house light illuminated, and a tone was generated.
  • Tissue samples were homogenized in 10 mM HEPES, 10 mM NaCl, 1 mM KH 2 PO 4 , 5 mM NaHCO 3 , ImM CaCl 2 , 0.5 mM MgCl 2 , 5 mM EDTA and the following protease inhibitors (PI): ImM phenylmethylsulfonylfluoride, 10 mM benzamidine, 10 ⁇ g/ml aprotinin, 10 ⁇ g/ml leupeptin, and 1 ⁇ g/ml pepstatin and centrifuged using a Beckman Coulter SW55Ti swinging bucket rotor at 5333 g for 5 min.
  • PI protease inhibitors
  • Protein concentrations were calculated using the bicinochoninic acid protein assay kit (Pierce, Rockford, IL) and diluted in Laemmli sample buffer to achieve the equivalent final protein concentrations. Five micrograms of protem were loaded into 10% sodium dodecyl sulfate-polyacrylamide gel, electrophoresed and transferred to nitrocellulose by electroblotting (30V, overnight at 4°C) in IX transfer buffer (Bio- Rad, Richmond, CA).
  • Nitrocellulose membranes were blocked in 0.5% w/v nonfat dry milk and 0.1% v/v Tween 20 in phosphate-buffered saline (pH 7.4, 0.12 M) for 1 hr at room temperature prior to being incubated with primary antibodies in blocking buffer (Bio-Rad) overnight at 4°C followed by secondary antibody for one hr at room temperature. Protein bands were visualized on a Kodak XAR-5 film with enhanced chemiluminescence (ECL plus, Amersham Pharmacia Biotech).
  • Primary antibodies were as follows: mouse monoclonal antibodies directed against NMDARI (Chemicon International, Temecula, CA) and rabbit polyclonal antibodies directed against NMDAR2A, NMDAR2B, NMDAR3A, NMDAR3B, GluRl, GluR2/3, GluR4, GluR5, GluR6/7, and KA2 (Upstate Biotechnology Cell Signaling Systems). Equal protein loading was confirmed by stripping the blots and re-probing them with a monoclonal /3-tubulin antibody (Upstate Biotechnology Cell Signaling Systems, Waltharn, MA; 1 :5000 v/v) followed by incubation with secondary antibody and visualization as described above.
  • the BINGE group self-administered approximately 344.4 ( ⁇ 48.3) infusions/172.2 ( ⁇ 24.2) mg of cocaine and the WITHDRAWAL group self- administered 359.5 ( ⁇ 48.0) infusions/179.8 ( ⁇ 24.0) mg of cocaine.
  • NMDARI was most abundant in the hippocampus, followed by the PFC, the NAc and striatum (HIPP>PFC>NAc, striatum» VTA, SN). There were no apparent differences in abundances of NMDAR2A, 2B, or 3 A among the hippocampus, PFC, NAc and striatum while these subunits were in low abundance in the VTA and SN.
  • the NMDAR3B subunit appeared to be most abundant in the PFC and striatum followed by the hippocampus and NAc. Due to the paucity of protein from the VTA and SN, NR3A and 3B levels were not assessed. The abundances of GluRl, GluR2/3 and GluR4 were greater in the hippocampus and PFC than the NAc and striatum (HIPP, PFC>NAc, striatum>VTA, SN). GluR5 protein levels appeared to be equally abundant in all regions tested. For the kainate receptor subunits, GluR6/7 was most abundant in the NAc, striatum and PFC followed by the hippocampus, then the SN and VTA. In contrast, KA2 was most abundant in the NAc, moderately abundant in the striatum, PFC and hippocampus and least abundant in the VTA and SN.
  • NR3B Protein levels were significantly altered by cocaine in the striatum
  • GluRl levels were significantly greater during withdrawal compared with binge access.
  • GluRl levels in the SN were increased during binge access but returned to control levels during withdrawal.
  • striatum levels were significantly elevated during withdrawal only (Figure 4A).
  • GluR2/3 Protein levels were significantly altered in the VTA
  • NR2B and NR3B levels were increased in the striatum following binge cocaine self-administration and remained elevated above control levels following two weeks of withdrawal suggesting these changes may represent long-term alterations in NMDA receptor function following cocaine exposure, hi contrast, NR3 A levels in the striatum were increased following binge cocaine access but returned to control levels following two weeks of withdrawal.
  • withdrawal from cocaine increased NR2B above control and binge access levels whereas withdrawal levels of NR3A were decreased compared to control and binge access levels.
  • the subunit is phosphorylated by PKA, PKC and possibly by CamKII which cause increased Ca 2+ influx tlirough the activated receptor leading to the slow onset of the EPSP.
  • NRl subunits may lead to enhanced Ca 2+ influx in dopamine neurons resulting in hyperexcitability of these cells.
  • NR3A alterations may provide an alternative means to increase intracellular Ca 2+ levels without altering the number of receptors.
  • GluRl and GluR2/3 levels in the VTA were significantly increased during withdrawal compared with binge access.
  • GluR2/3 levels were increased following withdrawal from cocaine compared with binge access, an effect also observed in human cocaine overdose victims.
  • Significant upregulation of GluR2/3 and GluR4 in the PFC were observed following withdrawal compared with control and binge access levels.
  • Kainate receptor subunits GluR6 and KA2 exhibited similar moderate to strong immunoreactivity in the
  • GluR5 levels were moderate to strong in the PFC and were similarly expressed in the NAc, striatum, VTA and SN. Interestingly, GluR5 levels were expressed at considerably higher levels in the rat compared with human VTA. Unlike NMDA and AMPA receptor subunits, the physiological function of kainate receptors remains unclear; however, they appear to be important for controlling Ca 2+ influx through the kainate ionophore in different pathological states, such as cocaine addiction (Paschen and Djuricic 1994).
  • GluR5, GluR6/7 and KA2 kainate receptor subunit levels were regulated in a region specific manner.
  • GluR5 and GluR6/7 immunoreactivities were decreased during binge access, whereas binge access increased GluR5, GluR6/7 and KA2 in the SN compared with withdrawal levels.
  • binge access increased GluR5, GluR6/7 and KA2 in the SN compared with withdrawal levels.
  • the kainate receptor subunits in the striatum were significantly upregulated during withdrawal compared with control and binge access levels.
  • the present study demonstrated the regional and subunit-specific changes in iGluR protein expression following binge cocaine self-administration and withdrawal. Theseresults provide a significant addition to the knowledge of altered glutamatergic function induced by chronic cocaine self-administration and withdrawal.
  • the study is unique in that it is the first study to assess alterations of multiple iGluR subtypes in various brain regions following binge cocaine self-admimstration and withdrawal. Such changes may be related to behaviors associated with withdrawal such as decreased locomotion, increased anxiety and behavioral sensitization and other enduring effects and may be an important mechanism by which cocaine exerts long- term effects on the mesolimbic dopamine system.

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Abstract

The present invention provides compositions and methods useful in the diagnosis and treatment of addictive disorders including cocaine addiction. Target genes and their expression products are identified based on their differential expression in subjects affected by addictive disorders in comparison with control subjects. In another aspect, the invention also provides methods for evaluating candidate drugs to predict their therapeutic efficacy. The invention also provides methods for predicting whether a compound will be addictive. Compositions of the invention include arrays, computer-readable mediums, and kits for use in the methods of the invention.

Description

MOLECULAR CORRELATES OF COCAINE ADDICTION AND METHODS FOR THEIR USE
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made in part with U.S. Government support under National Institute on Drug Abuse (DA13772 and DA15941). The U.S. Government may have certain rights in this invention.
FIELD OF THE INVENTION The present invention relates generally to molecular correlates of drug addiction and more specifically to the identification of molecular targets for use in diagnosing and treating addictive disorders.
BACKGROUND OF THE INVENTION In 1998, the National Household survey on Drug Abuse estimated that 1.8 million American were current cocaine users, 595,000 frequent users, and 2.4 million occasional users. With no significant decreases since 1992, these figures represent a persistent problem. Understanding the neural mechanisms that mediate cocaine abuse is critical for improving existing therapies and developing new treatment therapies. Chronic cocaine use results in particular intense euphoria and persistent drug dependence. In humans, the propensity to use cocaine is influenced by both positive (euphoric, pleasurable effects) and negative (withdrawal, depressed mood states, and drug cravings) consequences, including the development of neuroadaptive changes in specific brain regions (Gawin (1991) Science 251:1580-1586).
The characteristics of the addictive disorder although specific to cocaine itself, may generalize to other drag dependencies. Current understanding on the neuroadaptive processes stemming from chronic cocaine administration is based largely on rodent models of human drug taking, such as intravenous self- administration. However, reliance on such models has failed to yield useful pharmacotherapies for human cocaine addiction. Although the behavioral utility of rodent models for studying addictive processes is acknowledged, the biological utility of such models is questionable based on the anatomical and molecular complexity of the primate brain.
Accordingly, there remains a need for the identification of molecular targets in the diagnosis and treatment of cocaine addiction.
SUMMARY OF THE INVENTION The present invention provides methods for identifying genes and their expression products as targets in the diagnosis and treatment of addictive disorders. The targets are identified based on their differential expression in subjects affected by an addictive disorder in comparison with control subjects. Genes whose expression level is significantly increased or significantly decreased in subjects affected by an addictive disorder in comparison with control subjects are identified, along with their expression products, as screening targets for use in methods of identifying drugs for treating addictive disorders.
The invention provides an improved method for screening for therapeutic compounds for use in treating addictive disorders, where the improvement comprises the use of a target identified by the method described above. In another aspect, the present invention provides methods for producing an expression profile having values representing the expression levels of genes whose expression is correlated with addictive disorders. The method comprises determining the expression level of one or more genes in a sample from subjects affected by an addictive disorder, determining the expression levels of the same genes in control subjects, and identifying genes whose expression level is significantly increased or significantly decreased in subjects affected by the addictive disorder in comparison with control subjects.
The present invention also provides methods for predicting whether a compound will be addictive in humans. The methods comprise the steps of providing a reference expression profile associated with an addictive disorder, where the expression profile comprises values representing the expression level of one or more genes whose expression is associated with the addictive disorder in subjects affected by the addictive disorder, providing a test expression profile comprising values representing the expression level of said one or more genes in a sample comprising cells that been contacted with the compound; and determining whether said test expression profile shares sufficient similarity to said reference profile. When the test expression profile is sufficiently similar to the reference profile, it is predicted that the compound will be addictive in humans.
The invention identifies genes that are differentially expressed in cocaine overdose victims in comparison with control subjects, and provides a method of screening for therapeutic compounds for use in treating addictive disorders using these genes. The method comprises screening for modulators of genes that are differentially expressed in cocaine overdose victims in comparison with control subjects. Reports have shown that common molecular pathways, including the stimulation of dopamine transmission in the limbic system, underlie the addictive nature of a number of drugs including opiates, ethanol, nicotine, amphetamine, and cocaine. See, for example, Koob and Nester (1997) Journal ofNeuropsychiatry and Clinical Neurosciences 9:482-497; Wise and Bozarth (1987) Psychological Review 94:469-492; Di Chiara and Imperato (1988) Proc. Natl. Acad. Sci. U.S.A. 85:5274- 5278; Nestler (1997) Current Opinion in Neurobiology 7:713-719; and Self and Nestler (1995) Annual Review ofNeuroscience 18:463-495; each of which are herein incorporated by reference. Accordingly, genes having differential expression in cocaine overdose victims are targets for the diagnosis and treatment of a number of addictive disorders.
In another aspect, the invention provides methods for evaluating a candidate drug to determine whether it will have therapeutic efficacy in treating an addictive disorder. The method comprises the steps of determining the expression level of one or more genes in a first sample from a subject affected by an addictive disorder prior to treatment with the candidate drug, wherein expression of said one or more genes is associated with the addictive disorder; determining the expression level of said one or more genes in a second sample from the subject following treatment with the candidate drug, and comparing the expression level measured before and after the treatment to determine the change in the expression level of said genes following treatment with the candidate drug. If the drug results in a change in the expression levels of the genes associated with the addictive disorder, it results in a prediction that the candidate drug will have therapeutic efficacy in treating the disorder. Compositions for use in the methods of the invention are also provided. Compositions of the invention include an array comprising a substrate having a plurality of addresses, where each address has disposed thereon a capture probe that can specifically bind a nucleic acid molecule that is differentially expressed in subjects affected by cocaine addiction and a computer-readable medium having a digitally-encoded expression profile comprising one or more values representing the expression levels of a gene that is differentially expressed in cocaine overdose victims.
Compositions of the invention also include a kit for predicting whether a compound will be addictive in humans and a kit for evaluating the therapeutic efficacy of a candidate drug. The kits comprise an array having multiple addresses, wherein each address has a capture probe that can specifically bind a nucleic acid molecule that is differentially expressed in subjects affected by an addictive disorder; and a computer-readable medium having a digitally-encoded expression profiles having values representing the expression of a nucleic acid molecules detected by the array.
The methods of the invention have been applied to cocaine addiction to identify candidate targets for improving currently available treaments. Accordingly, one aspect of the invention is the use of the identified target genes in drug screening for drugs to treat addictive disorders.
DESCRIPTION OF THE FIGURES Figures 1A and IB: A. Ionotropic glutamate receptor subunit protein levels in VTA and 1-SN of cocaine overdose victims. Membrane fractions were isolated as described in the Experimental Procedures and 10 mg were separated on 10% SDS- PAGE to assess glutamate receptor subunit immunoreactivity. Data are expressed as mean (± S.E.M.) of the percent of control values per amount of protein loaded. Asterisks indicate a significant difference (PO.05). B. Representative bands from two cocaine overdose victims (+) and two control subjects (-) for each subunit.
Figures 2 A and 2B: A. CREB protein levels in the cytosolic and nuclear fragments of VTA and 1-SN in cocaine overdose victims. Cytoplasmic and nuclear fractions were isolated as described in the Experimental Procedures and 15 mg were separated on 10% SDS-PAGE to assess CREB immunoreactivity. Phospho-CREB immunoreactivity was not detectable in either the cytoplasmic or nuclear fractions. Data are expressed as mean (± S.E.M.) of the percent of control values. * indicate significant difference (P<0.05). B. Representative bands from two cocaine overdose victims (+) and two control subjects (-).
Figures 3 A and 3B: A. Levels of G-protein subunits in VTA and 1-SN of cocaine overdose victims. Cytoplasmic fractions were isolated as described in the Methods and 10 mg were separated on 10% SDS-PAGE to assess G-protein subunit immunoreactivity. Data are expressed as mean (± S .E.M.) of the percent of control values per amount of protein loaded. Asterisks indicate a significant difference (P<0.05). B. Representative bands from two cocaine overdose victims (+) and two control subjects (-).
Figure 4. Comparisons of ionotropic glutamate receptor immunoreactivity in brain regions following limited and binge cocaine self-adminstration and withdrawal. Post hoc comparisons reveal statistically significant differences aXp<0.05. C, control; B, binge; W, withdrawal.
DETAILED DESCRIPTION OF THE INVENTION
The present inventions now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown. Indeed, these inventions may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout.
Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.
The present invention demonstrates changes in gene expression levels in neuronal populations in subjects affected by an addictive disorder in comparison with control subjects. Changes in gene expression levels were detected in human postmortem samples, providing direct evidence of addiction-associated changes in gene expression in human subjects. Changes in gene experssion levels were also detected follwing binge cocaine self administration in rats. The identification of genes that are differentially expressed in subjects affected by addictive disorders provides methods for identifying genes and their expression products as targets in the diagnosis and treatment of addictive disorders. According to the invention, genes whose expression level is significantly increased or significantly decreased in subjects affected by an addictive disorder in comparison with control subjects are identified, along with their expression products, as screening targets for use in methods of identifying drugs for treating addictive disorders.
The methods of identifying genes whose expression is correlated with addiction provide an improvement in a method for screening for therapeutic compounds for use in treating addictive disorder. The improvement comprises the use of a target identified by the method described above. The identification of genes whose expression is correlated with addiction is also in creating expression profiles correlated with addiction. Accordingly, in another aspect, the present invention provides methods for producing an expression profile having values representing the expression levels of genes whose expression is correlated with addictive disorders. The method comprises determining the expression level of one or more genes in a sample from subjects affected by an addictive disorder, determining the expression levels of the same genes in control subjects, and identifying genes whose expression level is significantly increased or significantly decreased in subjects affected by the addictive disorder in comparison with control subjects. The invention also encompasses methods for predicting whether a compound will be addictive in humans. The methods comprise the steps of providing a reference expression profile associated with an addictive disorder, where the expression profile comprises values representing the expression level of one or more genes whose expression is associated with the addictive disorder in subjects affected by the addictive disorder, providing a test expression profile comprising values representing the expression level of said one or more genes in a sample comprising cells that been contacted with the compound; and determining whether said test expression profile shares sufficient similarity to said reference profile. When the test expression profile is sufficiently similar to the reference profile, it is predicted that the compound will be addictive in humans.
The invention identifies genes that are differentially expressed in cocaine overdose victims in comparison with control subjects, and provides a method of screening for therapeutic compounds for use in treating addictive disorders using these genes. The method comprises screening for modulators of genes that are differentially expressed in cocaine overdose victims in comparison with control subjects.
In another aspect, the invention provides methods for evaluating a candidate drug to determine whether it will have therapeutic efficacy in treating an addictive disorder. The method comprises the steps of determining the expression level of one or more genes in a first sample from a subject affected by an addictive disorder prior to treatment with the candidate drag, wherein expression of said one or more genes is associated with the addictive disorder; determining the expression level of said one or more genes in a second sample from the subject following treatment with the candidate drag, and comparing the expression level measured before and after the treatment to determine the change in the expression level of said genes following treatment with the candidate drug. If the drug results in a change in the expression levels of the genes associated with the addictive disorder, it results in a prediction that the candidate drug will have therapeutic efficacy in treating the disorder.
Accordingly, the present invention provides methods for predicating the therapeutic efficacy and the potential for addiction of a candidate drug. By "therapeutic efficacy" it is intended the ability of the drag to alleviate (e.g., mitigate, decrease, reduce) at least one of the symptom associated with the condition to be treated. By an "addictive" compound , it is intended a compound that creates a certain degree of euphoria in the subject and has a strong potential for addiction.
Compositions for use in the methods of the invention are also provided. Compositions of the invention include an array comprising a substrate having a plurality of addresses, where each address has disposed thereon a capture probe that can specifically bind a nucleic acid molecule that is differentially expressed in subjects affected by cocaine addiction and a computer-readable medium having a digitally-encoded expression profile comprising one or more values representing the expression levels of a gene that is differentially expressed in cocaine overdose victims.
Compositions of the invention also include a kit for predicting whether a compound will be addictive in humans and a kit for evaluating the therapeutic efficacy of a candidate drug. The kits comprise an array having multiple addresses, wherein each address has a capture probe that can specifically bind a nucleic acid molecule that is differentially expressed in subjects affected by an addictive disorder; and a computer-readable medium having a digitally-encoded expression profiles having values representing the expression of a nucleic acid molecules detected by the array.
Expression Levels and Expression Profiles
As used herein, an "expression level" or "level of expression" is a value that corresponds to a measurement of the relative or absolute abundance of a gene expression product. Such values may include measurements of RNA levels or protein abundance. Thus, an expression level can be a value that reflects the transcriptional state or the translation state of a gene.
The transcriptional state of a sample includes the identities and abundance of the RNA species, especially mRNAs present in the sample. The transcriptional state can be conveniently determined by measuring transcript abundance by any of several existing gene expression technologies. Translational state includes the identities and abundance of the constituent protein species in the sample.
As used herein, an "expression profile" comprises one or more values corresponding to a measurement of the relative or absolute abundance of a gene expression product. Such values may include measurements of RNA levels or protein abundance. Thus, the expression profile can comprise values representing the measurement of the transcriptional state or the translational state of the gene. See, U.S. Pat. Nos. 6,040,138, 5,800,992, 6,020135, 6,344,316, and 6,033,860, which are hereby incorporated by reference in their entireties. The transcriptional state of a sample includes the identities and relative abundance of the RNA species, especially mRNAs present in the sample. Preferably, a substantial fraction of all constituent RNA species in the sample are measured, but at least a sufficient fraction to characterize the transcriptional state of the sample is measured. The transcriptional state can be conveniently determined by measuring transcript abundance by any of several existing gene expression technologies. Translational state includes the identities and relative abundance of the constituent protein species in the sample.
In some embodiments, the expression profiles of the present invention are generated from samples from subjects affected by an addictive disorder. An
"addictive disorder" as used herein is a disorder involving a pattern of compulsive drag use characterized by a continued craving for a drug and the need to use the drug for effects other than pain relief. Examples of addictive disorders of the present invention include, but are not limited to, addiction to cocaine, addiction to opiates (e.g., heroin, methadone, morphine, oxycodone), addiction to stimulants
(amphetamines, methamphetamine), hallucinogens (LSD, MDMA), delta-9- tetrahycrocannabinol (THC), alcohol, nicotine, benzodiazepines, and/or barbiturates. The samples from the subject affected by an addictive disorder used to generate the expression profiles of the present invention can be derived from a variety of sources including, but not limited to, single cells, a collection of cells, tissue, or cell culture. The tissue or cell source may include a post-mortem sample, cell culture, or a single cell.
In selecting a sample, the percentage of the sample that constitutes cells having differential gene expression in addictive disorders should be considered. Samples may comprise at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% cells having differential expression in addictive disorders, with a preference for samples having a higher percentage of such cells. In some embodiments, these cells are neuronal cells. In specific embodiments, these cells are ventral tegmental area cells or lateral substantia nigra cells. In some embodiments the sample is of human origin, while in other embodiments it is derived from a non- human model system for addictive disorders. Examples of model systems include rodent model (e.g. self- admimstration models, conditioned place preference models, drag discrimination models, intracranial self-stimulation models, or locomoter activity models) and non-human primate models (e.g., rhesus monkey, squirrel monkey, baboon) for self-administration or drug discrimination. Examples of such model systems are described, for example, in Hemby et al. (1997) Neuropharmcological basis of drag reinforcement, in Drug Addiction and its Treatment: Nexus of
Neuroscience and Behavior, Johnson and Roache, Eds. Raven Press, NY, pp. 137- 169); Hemby (1999) Current Psychiatry Reports 1:159-165; Koob and Goders (1989) Neuroanatomical substrates of drug self-administration, in The Neuropharmcological Basis of Reward, Liebman JM and Cooper SJ eds.) pp. 214-263, Clarendon Press, Oxford; Koob and Nester (1997) Journal ofNeuropsychiatry and Clinical
Neurosciences 9:482-497; and Wise and Bozarth (1987) Apsychomotor stimulant theory of addiction. Psychological Review 94:469-492; each of which is herein incorporated by reference in its entirety.
The present invention also provides for measuring the level of gene in expression in control subjects. The control subjects of the invention are subjects that are not affected by drug addiction. In some embodiments, the control subjects matched to the test subjects affected by the addictive disorder by ensuring that both the control subject and the test subject are of a similar age. Where appropriate, other characteristics may also be matched between the control and test subjects such as race and sex. Matching characteristics of the test and control subjects reduces the likelihood that gene expression changes will results from a factor other than a difference in the use of addictive substances.
In some embodiments of the present invention, the expression profiles comprise values representing the expression levels of genes that are differentially expressed in subjects affected by an addictive disorder in comparison with control subjects. The term "differentially expressed" as used herein means that the measurement of a cellular constituent varies in two or more samples. The cellular constituent may be upregulated in a sample from a subject having one physiologic condition in comparison with a sample from a subject having a different physiologic condition, or down regulated in a sample from a subject having one physiologic condition in comparison with a sample from a subject having a different physiologic condition. Thus, the expression level of the gene may be significantly increased or significantly decreased in subjects affected by an addictive disorder in comparison with control subjects. By a "significant" change in expression level, it is intended a change in expression level that is statistically significant. A statistical test may be used to test whether a change in expression level measured for a gene after treatment is more likely to result from an actual change in the expression of the gene rather than from any variability present in the experimental system.
The present invention provides groups of genes that are differentially expressed in cocaine overdose victims in comparison with age-matched control subjects. These genes were identified based on gene expression levels for 8700 probes in 8 post-mortem tissue samples from cocaine overdose victims and 8 post- mortem tissue samples from control subjects, and on mRNA and protein levels for candidate genes (Table 2). Values representing the expression levels of the nucleic acid molecules detected were analyzed to identify those that were significantly up- or down-regulated in cocaine overdose victims in comparison with control subjects. See the Experimental section. The present provides genes with a differential level of expression in cocaine overdose victims compared to control subjects. These differentially expressed genes are selected from the genes shown in Tables 3, 4, and 5. These genes and their expression products are useful as markers to detect the presence of an addictive disorder in a patient. The present also provides glutamate receptor subunits with a differential level of expression in rats following binge access to cocaine in comparison with controls. The genes encoding these differentially-expressed receptor subunits are shown in Table 6. These genes and their expression products are useful as markers to detect the presence of an addictive disorder in a patient, and as screening targets for compounds to treat addicitive disorders.
Each expression profile of the invention contains a sufficient number of values such that the profile can be used to distinguish samples correlated with addictive disorders from control samples. In some embodiments, the expression profiles comprise only one value. In other embodiments, the expression profile comprises more than one value corresponding to a differentially expressed gene, for example at least 2 values, at least 3 values, at least 4 values, at least 5 values, at least 6 values, at least 7 values, at least 8 values, at least 9 values, at least 10 values, at least 11 values, at least 12 values, at least 13 values, at least 14 values, at least 15 values, at least 16 values, at least 17 values, at least 18 values, at least 19 values, at least 20 values, at least 22 values, at least 25 values, at least 27 values, at least 30 values, at least 35 values , at least 40 values, at least 45 values, at least 50 values, at least 75 values, at least 100 values, at least 125 values, at least 150 values, at least 175 values, at least 200 values, at least 250 values, at least 300 values, at least 400 values, at least 500 values, at least 600 values, at least 700 values, at least 800 values, at least 900 values, at least 1000 values, at least 1200 values, at least 1500 values, or at least 2000 or more values.
It is recognized that the diagnostic accuracy of the methods of the present invention will vary based on the strength of the correlation between the expression levels of the differentially expressed genes and the associated physiologic condition. When the values in the expression profiles represent the expression levels of genes whose expression is strongly correlated with the physiologic condition, it may be possible to use fewer number of values in the expression profile and still obtain an acceptable level of diagnostic or prognostic accuracy.
By a gene whose expression level is "correlated with" a particular physiologic condition, it is intended a gene whose expression shows a statistically significant correlation with the physiologic condition. The significance of the correlation between the expression level of a differentially expressed gene and a particular physiologic state may be determined by a statistical test of significance. Such methods are known in the art and examples are provided elsewhere herein. Methods for detemiining the strength of a correlation between the expression level of a differentially-expressed gene and a particular physiologic state are also reviewed in Holloway et al. (2002) Nature Genetics Suppl. 32:481-89, Churchill (2002) Nature Genetics Suppl. 32:490-95, Quackenbush (2002^ Nature Genetics Suppl. 32: 496-501 ; Slonim (2002) Nature Genetics Suppl.
32:502-08; and Chuaqui et al. (2002) Nature Genetics Suppl. 32:509-514; each of which is herein incorporated by reference in its entirety. Such methods may be used to select the genes whose expression levels have the greatest correlation with an addictive disorder in order to increase the predictive accuracy of the methods of the invention. Each value in the expression profiles of the invention is a measurement representing the absolute or the relative expression level of a differentially expressed genes. The expression levels of these genes may be determined by any method lαiown in the art for assessing the expression level of an RNA or protein molecule in a sample. For example, expression levels of RNA may be momtored using a membrane blot (such as used in hybridization analysis such as Northern, Southern, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids). See U.S. Patent Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which are expressly incorporated herein by reference. The gene expression monitoring system may also comprise nucleic acid probes in solution.
In one embodiment of the invention, microarrays are used to measure the values to be included in the expression profiles. Microarrays are particularly well suited for this purpose because of the reproducibility between different experiments. DNA microarrays provide one method for the simultaneous measurement of the expression levels of large numbers of genes. Each array consists of a reproducible pattern of capture probes attached to a solid support. Labeled RNA or DNA is hybridized to complementary probes on the array and then detected by laser scanning. Hybridization intensities for each probe on the array are determined and converted to a quantitative value representing relative gene expression levels. See, the Experimental section. See also, U.S. Pat. Nos. 6,040,138, 5,800,992 and 6,020,135, 6,033,860, and 6,344,316, which are incorporated herein by reference. High-density oligonucleotide arrays are particularly useful for determining the gene expression profile for a large number of RNA's in a sample.
In one approach, total mRNA isolated from the sample is converted to labeled cRNA and then hybridized to an oligonucleotide array. Each sample is hybridized to a separate array. Relative transcript levels are calculated by reference to appropriate controls present on the array and in the sample. See, for example, the Experimental section.
In another embodiment, the values in the expression profile are obtained by measuring the abundance of the protein products of the differentially-expressed genes. The abundance of these protein products can be deteπnined, for example, using antibodies specific for the protein products of the differentially-expressed genes. The term "antibody" as used herein refers to an immunoglobulin molecule or immunologically active portion thereof, i.e., an antigen-binding portion. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
The antibody can be a polyclonal, monoclonal, recombinant, e.g., a chimeric or humanized, fully human, non-human, e.g., murine, or single chain antibody. In a preferred embodiment it has effector function and can fix complement. The antibody can be coupled to a toxin or imaging agent.
A full-length protein product from a differentially-expressed gene, or an antigenic peptide fragment of the protein product can be used as an immunogen. Preferred epitopes encompassed by the antigenic peptide are regions of the protein product of the differentially expressed gene that are located on the surface of the protein, e.g., hydrophilic regions, as well as regions with high antigenicity. The antibody can be used to detect the protein product of the differentially expressed gene in order to evaluate the abundance and pattern of expression of the protein. These antibodies can also be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given drug. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance (i.e., antibody labeling). Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, biolummescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, b-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of biolummescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125L 131I, 35S or 3H.
Once the values comprised in the test expression profile and the reference expression profile or expression profiles are established, the subject profile is compared to the reference profile to determine whether the subject expression profile is sufficiently similar to the reference profile. Any method known in the art for comparing two or more data sets to detect similarity between them may be used to compare the subject expression profile to the reference expression profiles. To determine whether a subject expression profile shows "statistically significant similarity" or "sufficient similarity" to a reference profile, statistical tests may be performed to determine whether the similarity between the subject expression profile and the reference expression profile is likely to have been achieved by a random event. Examples of such a statistical tests are described in the Experimental section; however, any statistical test that can calculate the likelihood that the similarity between the subject expression profile and the reference profile results from a random event can be used. The accuracy of assigning a subject to a risk group based on similarity between an expression profile for the subject and an expression profile for the risk group depends in part on the degree of similarity between the two profiles. Therefore, when more accurate diagnoses are required, the stringency with which the similarity between the subject expression profile and the reference profile is evaluated should be increased. For example, in various embodiments, the p-value obtained when comparing the subject expression profile to a reference profile that shares sufficient similarity with the subject expression profile is less than 0.20, less than 0.15, less than 0.10, less than 0.09, less than 0.08, less than 0.07, less than 0.06, less than 0.05, less than 0.04, less than 0.03, less than 0.02, or less than 0.01.
Arrays, Computer-Readable Medium, and Kits The present invention provides compositions that are useful in the disclosed methods of identifying screening targets. These compositions include arrays comprising a substrate having a capture probes that can bind specifically to nucleic acid molecules that are differentially expressed in subjects affected by addictive disorders in comparison with control subjects. Also provided is a computer-readable medium having digitally encoded reference profiles useful in the methods of the claimed invention. The invention also encompasses kits comprising an array of the invention and a computer-readable medium having digitally-encoded reference profiles with values representing the expression of nucleic acid molecules detected by the arrays. These kits are useful for methods of evaluating the therapeutic efficacy of drags for treating addictive disorders, and methods for predicting whether a compound will be addictive in humans.
The present invention provides arrays comprising capture probes for detecting the differentially expressed genes of the invention. By "array" is intended a solid support or substrate with peptide or nucleic acid probes attached to said support or substrate. Arrays typically comprise a plurality of different nucleic acid or peptide capture probes that are coupled to a surface of a substrate in different, known locations. These arrays, also described as "microarrays" or colloquially "chips" have been generally described in the art, for example, in U.S. Patent. Nos. 5,143,854,
5,445,934, 5,744,305, 5,677,195, 6,040,193, 5,424,186, 6,329,143, and 6,309,831 and Fodor et al. (1991) Science 251:161-11, each of which is incorporated by reference in its entirety. These arrays may generally be produced using mechanical synthesis methods or light directed synthesis methods, which incorporate a combination of photolithographic methods and solid phase synthesis methods.
Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, e.g., U.S. Patent No. 5,384,261, incorporated herein by reference in its entirety for all purposes. Although a planar array surface is preferred, the array may be fabricated on a surface of virtually any shape or even a multiplicity of surfaces. Arrays may be peptides or nucleic acids on beads, gels, polymeric surfaces, fibers such as fiber optics, glass or any other appropriate substrate, see U.S. Pat. Nos. 5,770,358, 5,789,162, 5,708,153, 6,040,193 and 5,800,992, each of which is hereby incorporated in its entirety for all purposes. Arrays may be packaged in such a manner as to allow for diagnostics or other manipulation of an all-inclusive device. See, for example, U.S. Pat. Nos. 5,856,174 and 5,922,591 herein incorporated by reference.
The arrays provided by the present invention comprise capture probes that can specifically bind a nucleic acid molecule that is differentially expressed in cocaine overdose victims. In some embodiments, each capture probe in the array detects a nucleic acid molecule selected from the nucleic acid molecules designated in Tables 3, 4, and 5. The designated nucleic acid molecules include those differentially expressed in subjects affected by addictive disorders in comparison with control subjects.
The arrays of the invention comprise a substrate have a plurality of addresses, where each addresses has a capture probe that can specifically bind a target nucleic acid molecule. The number of addresses on the substrate varies with the purpose for which the array is intended. The arrays may be low-density arrays or high-density arrays and may contain 4 or more, 8 or more, 12 or more, 16 or more, 20 or more, 24 or more, 32 or more, 48 or more, 64 or more, 72 or more 80 or more, 96, or more addresses, or 192 or more, 288 or more, 384 or more, 768 or more, 1536 or more, 3072 or more, 6144 or more, 9216 or more, 12288 or more, 15360 or more, or 18432 or more addresses. In some embodiments, the substrate has no more than 12, 24, 48, 96, or 192, or 384 addresses, no more than 500, 600, 700, 800, or 900 addresses, or no more than 1000, 1200, 1600, 2400, or 3600 addressees.
The invention also provides a computer-readable medium comprising one or more digitally-encoded expression profiles, where each profile has one or more values representing the expression of a gene that is differentially expressed in a subject affected by an addictive disorder. In some embodiments, the digitally-encoded expression profiles are comprised in a database. See, for example, U.S. Patent No. 6,308,170. The database may be screened to identify genes to be used as targets in a screen for compounds useful for treating addictive disorders.
The present invention also provides kits useful for evaluating the therapeutic efficacy or potential for addition of compounds or candidate drugs. These kits comprise an array and a computer readable medium. The array comprises a substrate having addresses, where each address has a capture probe that can specifically bind a nucleic acid molecule that is differentially expressed in subjects affected by an addictive disorder in comparison with control subjects. The results are converted into a computer-readable medium that has digitally-encoded expression profiles containing values representing the expression level of a nucleic acid molecule detected by the array.
Methods of Screening and Therapeutic Targets The methods and compositions of the invention may be used to screen test compounds to identify therapeutic compounds useful for the treatment of addictive disorders. In one embodiment, the test compounds are screened in a sample comprising primary cells or a cell line representative of a neuronal cell type. After treatment with the test compound, the expression levels in the sample of one or more of the differentially-expressed genes of the invention are measured using methods described elsewhere herein. Values representing the expression levels of the differentially-expressed genes are used to generate a test expression profile. This subject expression profile is then compared to a reference profile associated with the addictive disorder represented by the sample to determine the similarity between the subject expression profile and the reference expression profile. Differences between the subject expression profile and the reference expression profile may be used to determine whether the test compound has therapeutic efficacy in treating an addictive disorder.
The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one- compound' library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145). Examples of methods for the synthesis of molecular libraries can be found in the art, for example in DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem. 37:1233. Libraries of compounds may be presented in solution (e.g., Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (U.S. Patent No. 5,223,409), spores (U.S. Patent No. 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science 249:404-406); (Cwiria et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 97:6378-6382); (Felici (1991) J Mol. Biol. 222:301-310).
Candidate compounds include, for example, 1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam et al. (1991) Nature 354:82-84; Houghten et al. (1991) Nature 354:84- 86) and combinatorial chemistry-derived molecular libraries made of D- and/or L- configuration amino acids; 2) phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang et al. (1993) Cell 12:161-118); 3) antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single chain antibodies as well as Fab, F(abj- 2, Fab expression library fragments, and epitope-binding fragments of antibodies); 4) small organic and inorganic molecules (e.g., molecules obtained from combinatorial and natural product libraries; 5) zinc analogs; 6) leukotriene A4 and derivatives; 7) classical aminopeptidase inhibitors and derivatives of such inhibitors, such as bestatin and a hamenine A and B and derivatives; 8) and artificial peptide substrates and other substrates, such as those disclosed herein above and derivatives thereof. The present invention discloses a number of genes that are differentially expressed in cocaine overdose victims in comparison with control subjects. These differentially-expressed genes are shown in Tables 3, 4, and 5. Because the expression of these genes is associated with drag addiction, these genes may play a role in the development of addiction. Accordingly, these genes and their gene products are potential therapeutic targets that are useful in methods of screening test compounds to identify therapeutic compounds for the treatment of addictive disorders.
The differentially-expressed genes of the invention may be used in cell-based screening assays involving recombinant host cells expressing the differentially- expressed gene product. The recombinant host cells are then screened to identify compounds that can activate the product of the differentially-expressed gene (i.e. agonists) or inactivate the product of the differentially-expressed gene (i.e. antagonists).
The following examples are offered by way of illustration and not intended to be limiting.
EXAMPLES Example 1: Differential Expression of Candidate Genes in Cocaine Overdose Victims A. Introduction Chronic cocaine use in humans and animal models is known to lead to pronounced alterations in neuronal function in brain regions associated with drag reinforcement. To evaluate whether alterations in gene expression in cocaine overdose victims are associated with specific dopamine populations in the midbrain, cDNA arrays and Western blotting were used to compare gene and protein expression patterns between cocaine overdose victims and age-matched controls in the ventral tegmental area (VTA) and lateral substantia nigra (1-SN). Array analysis revealed significant up- regulation of numerous transcripts in the VTA, but not 1-SN, of cocaine overdose victims including NMDARI , GluR2, GluR5 and KA2 receptor mRNA (PO.05). No significant alterations between overdose victims and controls were observed for GluRl, R3 or R4 mRNA levels. Correspondingly, Western blot analysis revealed VTA-selective up- regulation of CREB (PO.01) NMDARI (PO.01), GluR2 (PO.05), GluR5 (PO.01), and KA2 (PO.05) protein levels of cocaine overdose victims. The present results indicate that selective alterations of CREB and certain ionotropic glutamate receptor (iGluR) subtypes appear to be associated with chronic cocaine use in humans in a region specific manner. Moreover, since subunit composition determines the functional properties of iGluRs, the observed changes may indicate alterations in the excitability of dopamine transmission underlying long-term biochemical and behavioral effects of cocaine in humans.
Whereas animal models have advanced our understanding of the neurobiological basis of drug addiction, the evaluation of similar questions in human tissue are few, yet essential. Parallel investigations in human post-mortem tissue are important to determine whether biochemical changes observed and characterized in animal models are relevant to human drug abuse, as well as to identify novel changes that may be indicative of the human condition. The present study was undertaken to evaluate differences in gene expression in the VTA of cocaine overdose victims and age-matched, drag-free controls, hi addition, we evaluated gene expression in the 1-SN to provide a measure of brain region specificity. To this end, custom-designed macroarrays were used to simultaneously assess 81 genes and test the hypotliesis that functional classes of genes were differentially expressed between cocaine overdose victims and controls in the VTA. Computational analyses were used to partition the data into groups of clones with similar function to facilitate interpretation of these data to the relevance of cocaine addiction. Select genes that were differentially expressed were evaluated at the protein level by Western blot analysis. B. Experimental Procedures:
1. Subjects and Tissue:
Post-mortem human brain tissue was obtained at autopsy from 11 age-matched, drag-free control individuals (8WM, 1HM; IBM; WF; Age: 35.0 ± 2.3 yrs., PMI=13.2 hrs ± 0.6 hrs) and 10 cocaine overdose victims (8WM, 2BM; Age: 35.5 ± 2.2 yrs., PMI=12.3 hrs ± 0.9 hrs) by the University of Miami Brain Endowment Bank (Table 1). For protein analysis, tissue punches were dissected from the VTA and 1-SN of the contralateral hemisphere used for RNA analysis in eight cocaine overdose victims (34.4 ± 2.7 yr; 5WM, 3BM; PMI: 12.5 ± 1.1 hr) and seven age-matched, drug free controls (37 ± 2.7 yr; 6 WM, 1 WF; PMI: 12.6 ± 0.7 hr), representing a subset of subjects from whom tissue was used in the RNA experiments.
Figure imgf000023_0001
Figure imgf000024_0001
*Abbreviations: COC, cocaine; CE, cocethylene; COD: cocaine overdose; CTR: control; BE, benzylecognine; ND, not detectable; NA, not available; PMI, post-mortem interval; LC, lidocaine; EtOH, ethanol; DHPA: diphenhydramine
Gross and microscopic diagnostic neuropathologic examinations, which included examination of multiple cortical and subcortical regions, were performed in all cases and no neuropathological abnormalities relevant to mental status were found. All cases were retrospectively accrued based on toxicological data and circumstances surrounding the death, including review of prior arrest records and treatment admissions, as well as pathological indications (e.g., perforation of the nasal septum, needle track marks) were reviewed carefully before classifying a cocaine intoxication case. All cases were evaluated for common drugs of abuse and alcohol, and positive urine screens were confirmed by quantitative analysis of blood. Cocaine and benzoylecgonine concentrations in brain and blood were assessed using gas chromatography/mass spectroscopy as described previously in Hernandez et al. (1994) Forensic Science International 65: 149-156, herein incorporated by reference in its entirety.
Following removal, brains were photographed and cut into 1.5 cm coronal blocks. Brain tissue was cryopreserved using a procedure described in Hardy et al. 1983 J Neurochem. 40: 608-614; and Dodd et /. 1 Neurochemical Pathology 4, 177-198, both of which are herein incorporated by reference. One hundred mg punches were dissected from the blocks containing the VTA (posterior region of slab) and SN (anterior region containing the pars lateralis and the pars medialis) - one hemisphere for RNA and the contralateral hemisphere for protein analysis. Possible neuronal loss, ischemic cell changes, and reactive gliosis were assessed using semiquantitative ratings by the neuropathologists and found to be negligible in all cases used in the present study.
2. RNA isolation and amplification: Micro-FastTrack® brand RNA isolation kits (Invitrogen; Carlsbad, Cah" fornia) were used to isolate polyadenylated RNA from the VTA and 1-SN. mRNA yields ranged from 1 to 2 mg/100 ng of frozen tissue. Due to the amount of mRNA, samples from each subject were amplified using a modification of the aRNA amplification procedure described previously in Ginsberg et al. (2000) Ann Neurol. 48:77-87; Hemby et al. (2002) Archives of General Psychiatry 59:631-640; and Hemby et al. (2003) Journal of Comparative Neurology 456: 176-183, each of which is herein incorporated by refrence in its entireity. Oligo(dT)-T7- primer/promoter (500 ng) [AAACGACGGCCAGTGAATTGTAAT ACGACTCACTATAGGCGC(T)24] (SEQ ID NO:l) was hybridized to poly(A+)mRNA in the presence of 500 mM each of dNTPS and RNase free water for 30 minutes at 65°C, then quick cooled on ice for 5 minutes. Next, 10 mM DTT, IX first strand buffer and 20U RNAsin were added and samples were incubated at 42°C for 2 minutes as described in Van Gelder et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87:1663- 1667. One ml of reverse transcriptase (Superscript™ π, RNase H-; invitrogen; Carlsbad, California) was added to the solution and incubated at 42°C for 60 minutes. Following phenol/chloroform extraction and ethanol precipitation in the presence of 10 mg linear acrylamide, samples were re-suspended in RNase free water and heat denatured at 94°C for 5 minutes, then quick cooled. Next, IX second strand buffer, 250 mM each of dNTPs, 10U Klenow fragment and 10U RNase H were added to the sample and incubated at 16°C for 4-6 hrs as described in Van Gelder et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87:1663-1667. At the end of the incubation, 2.5 mM b-nictotanfmide adenine dinucleotide and 1000U E. coli DNA ligase were added to the solution and incubated at room temperature of 15 min, followed by the addition of 5U T4 DNA polymerase at room temperature for 15 min as described in Sambrook and Russell (2001) J Neurosci. 18:1848-1859. At the end of the incubation, 2.5 mM EDTA was added to stop the enzymatic reaction followed by phenol/chloroform extraction and ethanol precipitation. Following drop dialysis against RNase DNase free water for 15 minutes, purified cDNA templates were used to generate antisense RNA (aRNA). aRNA was synthesized using T7 RNA polymerase (EpiCentre Technologies; Madison, WI) in IX transcription buffer (Epicentre Technologies), 10 mM DTT, 250 mM each of NTPs and 20U RNAsin at 37°C overnight. Following phenol/chloroform extraction and ethanol precipitation, aRNA was reverse transcribed using Superscript II in the presence of IX 1st strand buffer, 250 mM each of dNTPs, 100 ng of random hexamers and 20U RNAsin incubated at 37°C for one hr. For second strand cDNA synthesis, samples were incubated with 10U Klenow fragment, 250 mM each of dNTPs, 500 ng of the aforementioned oligo dT-T7 primer-promoter, and 5U T4 DNA polymerase at 16°C overnight. Samples were phenol/chloroform extracted, ethanol precipitated and drop dialyzed for 15 min.
For the second round of amplification, reaction conditions were identical for the first round except for the inclusion of 30 mCi [a-33P]-UTP (2500 Ci/mmol; Amersham Pharmacia Biotech; Piscataway, NJ), 10 mM of UTP and 250 mM each of CTP, GTP and ATP. The aRNA procedure is a linear amplification process with minimal change in the relative abundance of the mRNA population in the native state of the neuron. mRNA can be reliably amplified from small amounts of fixed tissue including individual neurons and neuronal processes (Van Gelder et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87:1663-1667; Eberwine et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89:3010-3014; Ginsberg et al. 1999 Annals of Neurology 45:174-181; Hemby et al. (2002) Archives of General Psychiatry 59:631 -640; and Hemby et al. (2003) Journal of Comparative Neurology 456:176-183). Although there is a general decrease in mRNA levels in autopsied tissue, the relative abundance of gene expression remains unchanged (Castensson et al. (2000) Genome Research 10:1219-1229).
3. Macroarray Procedures
Reverse Northern Blots were prepared on nylon membranes containing candidate genes including dopamine receptors (e.g. Dl, D2, D4, D5 and DAT), G- protein subunits (ail, ai2, as, az, aq, ao, β, γl and γ2), glutamate receptor mRNAs (mGluR3, GRIA1-4, GRD 5, 7 and GRIN1), GABA A receptor subunits (αl, α2, βl, β3, γ2, δ, ε, and π), regulators of G-protein signaling (RGS 1-7, 9, 10, 12, 13, 16) and other transcripts (cannabinoid 1 receptor, cocame-amphetamine regulated transcript (CART), serotonin 2 and serotonin 3 receptors, and tyrosine hydroxylase). Inserts were amplified in 96 well plates using PCR with GF200 primers under the following conditions: 95°C for 5min (1 cycle); 95°C for 30 sec, 52°C for 45 sec, and 72°C for 2 min (40 cycles); and 72°C for 7 min (1 cycle). PCR samples were purified (Multiscreen™ PCR filter plate, Millipore, Bedford, MA) and aliquots were electrophoresed on a 1% agarose gel (IX TAE, 0.05% ethidium bromide) at 5V/cm for PCR band size verification. Gel images were captured by digital camera and archived. PCR product concentration was determined by spectrofluorometry (Gemini Microplate Spectrofluorometer, Molecular Devices, Sunnyvale, CA) using a 1:5000 dilution of SYBR 1 Green/TE and an aliquot of the PCR product. Values were compared to concentrations of known DNA standards for quantitation. 250 ng of each amplified insert was spotted on Nytran SuPerCharge ® nylon transfer membrane (Schleicher and Schuell, Keene, New Hampshire) using a 96 well dot blot apparatus (Schleicher and Schuell; Minifold I). DNA was crosslinked to the membrane by ultraviolet radiation.
Arrays were pre-hybridized with ULTRAhyb™ solution (Ambion; Austin, TX) in hybridization bottles for 1 hr at 42°C. Next, P-labelled aRNA probes from each subject were heat denatured for 5 min at 65°C, and then immediately added to their respective bottles and allowed to hybridize for 18-24 hrs at 42°C in a rotisserie hybridization oven. Following hybridization, membranes were washed twice with 2X SSC/0.1% SDS and 0.1X SSC/0.1% SDS for 20 min each at 42°C. Labeled hybridized products were detected using phosphorimager screens, and hybridization signal intensities were analyzed using ImageQuant™ software (Amersham Biosciences; Sunnyvale, CA).
4. Protein preparation and Western blot analysis Tissue samples were dounce homogenized in 10 mM HEPES, 10 mM NaCl, 1 mM KH2PO4, 5 mM NaHCO3, ImM CaCl2, 0.5 mM MgCl2, 5 mM EDTA and the following protease inhibitors (PI): ImMPMSF, lO mMbenzanήdine, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 1 mg/ml pepstatin and centrifuged at 7500 RPM for 5 min. Supernatant was removed and the pellet (nuclei and debris) was resuspended in 20 mM Tris HCl, 1 mM EDTA (pH=8.0) with Pis and centrifuged at 7500 RPM for 5 min. This procedure was repeated twice and the pellet was resuspended in the solution and stored at -20°C (nuclear fraction). Supernatant was centrifuged at 25,000 RPM for 30 min at 4°C. Next, the supernatant containing the cytosolic fragment was removed and stored at -20°C (cytosolic fraction). The pellet was re-suspended in 10 mM Tris
(pH=7.5), 300 mM sucrose, 1 mM EDTA (pH=8.0), 0.1% NP40 and Pis and centrifuged at 5000 RPM for 5 min at 4°C. The supernatant was discarded and the pellet was resuspended in the buffer and washed three times before re-suspension in the buffer and Pis and storing the samples at -20°C (membrane fraction). Protein concentrations of samples were calculated using a bicinochoninic acid reagent protein assay kit (Pierce, Rockford, EL) and then the samples were diluted in Laemmli sample buffer to achieve the same final protein concentration. Identical amounts of proteins were loaded into a gel electrophoresis apparatus, subjected to sodium dodecyl sulfate-polyacrylamide (10%) gel electrophoresis (Bio-Rad, Richmond, CA) and transferred to nitrocellulose by electioblotting (30V, overnight at 4°C) in IX transfer buffer (Bio-Rad). Nitrocellulose membranes were blocked in 0.5% nonfat dry milk and 0.1% Tween 20 in phosphate-buffered saline (pH 7.4, 0.12 M) for 1 hr at room temperature prior to being incubated with primary antibodies in blocking buffer (Bio- Rad) overnight at 4°C followed by secondary antibody for one hr at room temperature. Protein bands were visualized on a Kodak XAR-5 film with enhanced chemiluminescence (ECL Plus™, Amersham Pharmacia Biotech). Primary antibodies were as follows: mouse monoclonal antibodies directed against NMDARI (Chemicon International, Temecula, CA) and GluR5 (Upstate Biotechnologies, Waltham, MA); rabbit polyclonal antibodies directed against GluRl, GluR2/3, KA2, G s, Gail/2, Gβ, CREB , phospho-CREB (Upstate Biotechnologies) and FRA-2 (L- 15 ; Santa Cruz
Biotechnology, Santa Cruz, CA). Secondary antibodies were HRP-conjugated anti-rabbit IgG and HRP-conjugated anti-mouse IgG (Upstate Biotechnologies). Protein abundances were calculated by optical densitometry with a Scan Jet 2200C and imported into NIH Image 1.61 software. Film background was subtracted from the optical density values to give the optical density value for a single subject. All assays were conducted under conditions in which densitometric signal intensity was linear with protein concentration as determined in preliminary experiments. 5. Data Analysis
Specific signal (minus background) of probe bound to each PCR product was expressed as a ratio of the total hybridization intensity of spots of the custom-designed array, thereby minimizing variations due to differences in the specific activity of the probe and the absolute quantity of probe present (Ginsberg et al. (2000) Ann Neurol. 48:77-87; Hemby et al. (2002) Archives of General Psychiatry 59:631-640; and Hemby et al. (2003) Journal of Comparative Neurology 456: 176-183). Array data were analyzed using a two way ANOVA with group and anato ical region as the fixed effects and normalized hybridization intensity as the dependent measure. Post hoc analyses were conducted as needed using Tukey's Test and the null hypothesis was rejected when P<0.05. Western blot data were analyzed using t-tests comparing the band intensities between cocaine overdose victims and controls for each respective protein. Data were graphically depicted as percent of the controls for each respective protein. Null hypotheses were rejected when P<0.05.
C. Results
1. Subject data
There was no significant difference in age between cocaine overdose victims and age-matched, non-drug controls (t=-0.156, df=19, P=0.877), post-mortem interval (t=0.847, df=19, P=0.408), or brain pH (t=0.431, df=17, P=0.672) indicating these factors did not significantly influence the observed changes in gene or protein expression. Furthermore, neuroadaptive changes in the human brain post-mortem reflect chronic cocaine abuse, since death in a naive user is a rare occurrence, and the cohort of post-mortem subjects have many surrogate measures of chronicity (Ruttenber et al. 1991 Journal of Forensic Science. 42:25-31).
2. Gene Expression
Custom-designed macroarrays containing 81 cDNAs were used to evaluate gene expression changes in the VTA and 1-SN of cocaine overdose victims and controls. The arrays contained a variety of transcripts including iGluR subunits (n=8), dopamine signaling (n=7), GAB A transcripts (n=10), G-protein subunits (n=14), GTPase/RGS proteins (n=16), mRNA processing transcripts (n=8), cell growth/death transcripts (n=5), and others (n=l 3). Receptors. There was a significant effect of cocaine overdose on glutamate receptor subunit mRNA expression in the VTA [F(l,209)=18.541; PO.001] with a significant interaction between TRANSCRIPT and GROUP [F(l,9)=2.311; PO.017]. Post hoc analysis revealed a significant increase in NMDARI, GluR2, GluR5 and KA2, but not GluRl, GluR3 or GluR4 glutamate receptor subunits or the mGluR3 receptor mRNAs in the VTA of cocaine overdose victims (Table 2). mGluR3 and GluR3 were in low abundance in the VTA. Furthermore, no significant differences were observed in glutamate receptor mRNA expression in the 1-SN between cocaine overdose victims and controls. There was no significant difference in mRNA abundance for the various dopamine signaling transcripts including receptor subtypes (Dl, D4, and D5), the dopamine transporter, tyrosine hydroxylase and dopa decarboxylase between cocaine overdose victims and controls in either the VTA [F(l, 146)0.179, P .679] or 1-SN [F(l, 139)0.135, P0.714]. There was a trend towards significance in GABA signaling transcripts including GABA A receptor subunits (αl, α2, βl, β3, γ2, δ, ε, π) and glutamic acid decarboxylases (GAD) 65 and 67 in the VTA [F(l,230)=3.843, PO.051]; however, there was no significant difference in these transcripts between the groups in the 1-SN [F(l, 219)=1.980, P .161]. Analysis of other neurotransmitter signaling transcripts (CART, serotonin receptor subtype 2C and 3, and cannabinoid receptor 1) revealed a significant GROUP by TRANSCRIPT interaction [F(l,4)=4.440, P .003) that was attributable to a down-regulation of CART in the VTA of cocaine overdose victims compared to controls (PO.05).
3. Signaling Cascade Transcripts Examination of a variety of G-protein subunit mRNAs (Gαl 1, Gαl5, Gαl,
Gail, Gai2, Gas, Gat, Gaz, Gaq, Gao, Gβl, and Gγl-4) revealed a significant
GROUP and TRANSCRIPT interaction [F(l,16)=1.863), PO.023] in the VTA that was attributable to a down-regulation of the Gbl subunit in cocaine overdose victims
(PO.05). Interestingly, there was a significant down-regulation of G-protein subunits in the 1-SN of cocaine overdose victims compared to controls [F(l,339)=4.589, PO.033], although there was no interaction.
GTPases and RGS proteins play an important role in regulating G protein function. In the VTA, there was a significant interaction of GROUP and TRANSCRIPT for RGS/GTPase mRNAs (regulator of G-protein signaling (RGS) 1-7, 9, 10, 12, 13, 16, Rho guanine nucleotide exchange factor 5 (timl), RAB7, member RAS oncogene family-like 1 (RAB7L1), and Ras homolog enriched in brain 2 (RHEB2)) [F(l,314)=2.514, PO.002] that was attributable to a significant up-regulation of RGS3 (PO.05) and down-regulation of RGS12 (PO.05) in cocaine overdose victims. In contrast, there was a significant difference in the overall abundance of these transcripts between cocaine overdose victims and controls in the 1-SN [F(l,298)=4.951, PO.027], but no significant interaction (Table 2).
4. mRNA processing and cell growth/death related transcripts
Analysis of several transcript encoding proteins related to mRNA processing (fos-related antigen 2 (FRA2); early growth response (EGR) 2 and 3; protein kinase interferon-inducible double stranded RNA dependent (PRKR); CUG triplet repeat RNA binding protein 2 (CUGBP2); RNA polymerase JJ-DNA directed- polypeptide C 33kDa (POLR2C); suppressor of RNA polymerase B7 (RNApH); TATA box binding protein- associated factor, RNA polymerase IT, H, 30kD (TBP-af30)) did not reveal any significant difference between cocaine overdose victims and controls in either the VTA or 1-SN. Likewise, there was no significant difference in the abundance of cell growth/death related transcripts (activity-regulated cytoskeleton-associated protein (arc); brain derived neurotrophic factor (BDNF); growth associated protein 43 (GAP43); alpha spectrin, non-erythrocytic 1 (a-fodrin); and BCL2-associated X protein (bax)) between the groups in either the VTA or 1-SN (Table 2).
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
5. Protein expression. Western blot analysis was performed on protein homogenates from individual subjects to assess the correlation of changes in mRNA levels with protein levels. From the gene expression analysis, proteins for NRl, GluRl, GluR2, GluR5, KA2, Gail, Gas, and Gβ were selected based on the availability of selective antibodies, hi addition, protein levels of cAMP responsive element binding protein (CREB), phosphorylated CREB, and FRA2 were evaluated based on their regulation in animal models of drug abuse (Carlezon et al. (1998) Science 282:2272-2275; Self et al. (1998) Journal of Neuroscience 18:1848-1859; Pliakas et al. (2001) Journal ofNeuroscience 21:7397- 7403). h membrane fractions, NRl immunoreactive protein was increased 111.2% (PO.05), GluR2/3 by 69.1% (PO.05), GluR5 by 507.9% (P .01), and KA2 by 72.1%
(PO.05) in the VTA of cocaine overdose victims, whereas GluRl showed a nonsignificant 50.3% increase in this region. Comparatively, these proteins were slightly increased in the 1-SN, albeit in a non-significant manner (NRl : 12.9%; GluRl : 56.1%; GluR2/3: 30.1%; GluR5: 13.4%; KA2: 50.4%). Aprevious study indicates that the aforementioned iGluR subunits are stable up to 18 hr PMI (Wang et al. (2000) Molecular Brain Research 80: 123-131), suggesting that the present results are not compromised by proteolysis.
Protein levels were increased 23.3% for Gαil/2 and 23.6% for Gas while Gβ levels were decreased 21.1% in the VTA of cocaine overdose victims compared to controls in cytosolic fragments. In the 1-SN, Gαil/2 was increased by 10%, whereas Gas was decreased by 15.1% and Gβ 10.1% in cocaine overdose victims compared to controls, all in a statistically non-significant manner.
Previous studies have hypothesized that adaptive changes induced by cocaine on the cAMP intracellular cascade can alter subsequent responsiveness of the drug (Carlezon et al. (1998) Science 282:2272-2275; Self et al. (1998) Journal of
Neuroscience 18:1848-1859; Pliakas et al. (2001) Journal ofNeuroscience 21:1391- 7403). Evaluation of CREB immunoreactive protein revealed a significant up-regulation (69.6%; PO.05) of CREB in the cytosolic fraction from the VTA, but not 1-SN, of cocaine overdose victims. There was a slight but statistically non-significant increase in CREB immunoreactivity in the nuclear protein fraction from the VTA (34.3%), whereas levels in the 1-SN were similar between cocaine overdose victims and controls. Phospho-CREB immunoreactivity was not detectable in either the cytosolic or nuclear fractions in either brain regions of cocaine overdose victims or controls (data not shown). There was no significant difference in FRA-2 protein levels between cocaine overdose victims and controls in either the VTA or 1-SN.
D. Conclusions
The results described above demonstrate the up-regulation of NRl, GluR2, GluR5 and KA2 glutamate receptor subunit mRNA and protein levels specific to the VTA in human cocaine overdose victims. Previous studies in rodents indicated that upregulation of iGluR subunits was associated with augmented dopamine-glutamate interactions in the mesolimbic pathway (White et al. (1995) Journal of Pharmacology & Experimental Therapeutics 273:445-454; White et al. (1995) Neurophysiological alterations in the mesocorticolimbic doapmine system during cocaine administration, in The Neurobiology of Cocaine Addiction, Hammer R. P., Jr., ed. pp 99-120, CRC Press, Boca Raton, FL; Zhang et al. (1997) Journal of Pharmacology & Experimental Tlerapeutics 281:699-706; White and Kalivas (1998) Drug & Alcohol Dependence 51:141-153; Wolf (1998); Vanderschuren and Kalivas (2000) Psychopharmacologia 151:99-120; and Giorgetti et al. (2001) Journal ofNeuroscience 21 :6362-6369. The present invention demonstrates that increased NRl protein levels in cocaine overdose victims may be induced by acute or sub-acute cocaine exposure or possibly reflect the last acute cocaine binge prior to death. Upregulation of mRNA and protein levels of GluR2, GluR5 and KA2 are indicative of chronic cocaine use and/or cocaine overdose in humans.
In the VTA and SN, GluRl -4 and NRl immunoreactivity was found to be localized almost exclusively on dopaminergic neurons (Paquet et al. 1997 J Neurosci. 17:1377-1396); however, less is known about the localization of kainate subunits in these regions. Glutamatergic afferents in the midbrain originate from the medial frontal cortex, subthalamic and pedunculopontine tegmental nuclei (Christie et al. (1985) Brain Research 333:169-172.; Sesack et al. (1989) Journal of Comparative Neurology 290:213-242; Sesack and Pickel (1992) Journal of Comparative Neurology 320:145- 160; and Lu et al. (1997) . Synapse 25:205-214. A recent study suggested prefrontal glutamatergic projections selectively synapse on mesoprefrontal dopamine neurons and mesoaccumbens GABA neurons in the VTA in rats (Carr and Sesack (2000); Journal of Neuroscience 20:3864-3873, although the topography in non-human primates and humans remains unknown. The presence of these subunits on dopamine neurons and the upregulation reported in the present study may provide a means by which to influence dopamine excitability in this region. Upregulation of the kainite preferring subunits GluR5 and KA2 in cocaine overdose victims and the probable localization of these subunits on dopamine neurons represent additional means by which increased Ca2+ influx may lead to hyperexcitability of VTA dopamine neurons. Interestingly, GluR2 subunit protein levels were increased in the hippocampus of alcoholics (Breese et al. (1995) Brain Research 674:82-90) and GluR5 mRNA and protein levels were increased in the dorsal prefrontal cortex of cocaine treated rats (Toda et al. (2002) Journal of Neuroehemistry 82, 1290-1299). Since extensive editing of GluR2 in the adult brain renders ionophores less permeable to Ca2+, the upregulation observed in the present study may represent a molecular compensation for the effects of increased Ca2+ influx generated by the combined upregulation of NRl, GluR5 and KA2 (Paschen et al. (1994) Journal of Neuroehemistry. 63:1596-1602). In summary, although the present invention is not bound by a particular mechanism, upregulation of the iGluR subunit mRNA and protein levels in the present study are likely attributable to changes in VTA dopaminergic neurons, suggest ng a neuroadaptive response to cocaine in these subjects. Dopamine receptor mRNAs were not differentially regulated as a function of cocaine overdose, opening the possibility that intracellular signaling mechanisms may also contribute to the altered function of these neurons. Neuroadaptations in the cAMP pathway have been implicated in mesolimbic brain regions as a function of cocaine exposure (Nestler et al. (1990) Journal of Neuroehemistry. 55:1079-1082; Terwilliger et al. (1991) Brain Research. 548:100-110; Striplin and Kalivas (1992) Brain Research. 579:181-186; Miserendino and Nestler (1995) Brain Research. 674:299-306; Carlezon et al. (1998) Brain Research. 674:299-306; Self et al. (1998) Journal ofNeuroscience. 18:1848-1859; Pliakas et al. (2001) Journal ofNeuroscience. 21:7397-7403). Assessment of various α, β, and γ G-protein subunits revealed only a significant decrease in Gβl subunit mRNA, but not protein, in the VTA of cocaine overdose victims. The discrepancy between Gβl mRNA and protein levels may be reflective of post-transcriptional, translational, or post-translational processing/degradation and or trafficking of the protein outside of the regions studied. In addition, decreased Gβ mRNA levels may manifest as down-regulated protein levels in axonal targets of the mesoaccumbens dopamine neurons as shown previously in cocaine-treated rats (Wang et al. (1991) Journal of N eur oscience 17:5993-6000. The present data contrast previous studies showing decreased ADP ribosylation and immunoreactivity Gαi and Gαo in the VTA of cocaine treated rats. However, a significant increase in CREB mRNA and protem levels in the cytosolic fraction and a trend towards significance in the nuclear fraction in the VTA of cocaine overdose victims was observed - the first demonstration of increased CREB protein levels in the VTA of either humans or animal models as a function of cocaine exposure. The length of the post mortem intervals for these subjects likely contributed to the inability to detect phosphorylated CREB imunoreactivity; precluding speculation on the role of elevated CREB levels in the present study. Although CREB is almost exclusively expressed in the nucleus, CREB protein has been identified in the cytoplasm by light and electron microscopy (Ferrer et al. (1996) Journal ofNeuroscience 16:274-282; Hermanson et al. (1996) Biochemical & Biophysical Research Communications 225:256-262 ; Suzuki etal. (1998) Molecular Brain Research. 61:69-77; Shaywiiz and Greenberg (1999) Annual Review of Biochemistry 68:821-861) as well as in posi-synaptic density fractions (Suzuki et al. (1998) Molecular Brain Research. 61:69-77) and dendriies (Crino et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95:2313-2318) leading to the hypothesis of nuclear translocation as a means of site-specific plasticity (Crino et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95:2313-2318; Suzuki et al. (1998) Molecular Brain Research. 61:69-11). hi the present study, statistical analysis indicated a trend towards significance in nuclear CREB (PO.07) in the VTA of cocaine overdose victims. In addition, the ability of the antibody to detect the antigen in the nucleus may be impaired by conformational changes in the protein due to dimerization, binding of the dimer to DNA, and/or masking of the antigen by CREB binding proteins or other transcriptional regulators. A more precise role of cytoplasmic CREB and the mechanism underlying elevated CREB levels remains to be deteπnined in animal models of the disease. The relevance of altered CREB levels in the present study supports several studies in rodents which indicated CREB levels were inversely proportional to the rewarding effects of the drug (Carlezon et al. (1998) Science. 282:2272-2275; Walters and Blendy (2001) Journal of
Neuroscience 21:9438-9444.. One manner in which CREB activity may be regulated is by Ca2+ influx via NMDA receptors, demonstrating a requirement for CREB-NMDA receptor interactions in dopamine-regulated gene expression (Konradi et al. (1996) Journal ofNeuroscience 16, 4231-4239). One of the transcripts regulated by CREB is CART (Dominguez et al. (2002)
Journal of Neuroehemistry 80:885-893). Discovered as a novel transcript in the striatum whose regulation was induced by acute cocaine (Douglass et al. (1995) Journal of Neuroscience 15:2471-2481), CART peptides were shown to be abundant in the NAc, VTA and SN as well as other brain regions (Smith et al. (1997) Synapse 27:90-94; Smith et al. (1999) Journal of Comparative Neurology 407:491-511; Hurd and Fagergren (2000) Journal of Comparative Neurology 425:583-598; and Dallvechia-Adams et al. (2002) Journal of Comparative Neurology 448:360-372. The present study is the first to demonstrate CART mRNA regulation by chronic cocaine use in humans.
The upregulation of RGS3 and RGS 12 mRNAs in the VTA of cocaine overdose victims represents another potential mechanism for cocaine-induced alterations in cell signaling. Previous studies have shown elevated RGS 2, 3 and 5 mRNA levels in the striatum by acute amphetamine administration (Burchett et al. (1999) Journal of Neuroehemistry 72:1529-1533) and RGS 2 mRNA levels by cocaine in the hippocampus, cortex, and striatum (Ingi etal. (1998) Journal oj "Neuroscience 18:7178- 7188). RGS proteins modulate/integrate G-protein and other intracellular signaling proteins, some of which reduce the duration of G-protein activation by increasing the rate of GTP hydrolysis. In vivo, RGS3 interacts with tlie G protein bg complex to accelerate the rate of recovery of N-type Ca2+ inhibition from Gbg thereby leading to increased Ca2+ conductance and potentially to increased excitability of VTA dopamine neurons.
Example 2: Microarray Analysis of Molecular Correlates of Cocaine Addiction The present example describes the identification of genes that are differentially expressed in the VTA of cocaine overdose victims in comparison with age-matched controls. Gene expression from the VTA of cocaine overdose victims (n=8) and age- matched controls (n=8) using high-density microarrays (UniGem V. 2.0, Incyte Pharmaceuticals, Inc) having capture probes for 8700 human transcripts. mRNA from each subject was isolated as described above in Example 1. Equivalent concentrations of mRNA from each subject within the cocaine overdose group or the control group were combined. Pooled samples were reverse transcribed and hybridized to the microarrays, and the resulting image was analyzed to determine gene expression levels. Pooling samples decreases the influence of individual variability and stresses the identification of those transcripts that are most highly regulated in the sample population. Hierarchical (degree of differential expression) and functional (protein classes, functional pathways, etc.) clustering of the data reveals a subset of genes that were assessed further by subsequent reverse Northern analysis. Using 1.8-fold differential expression as a cut-off for the hierarchical analysis, greater than 250 transcripts are down-regulated (Table 4) and greater than 1800 transcripts are up-regulated (Table 5) in the VTA from cocaine overdose victims.
Table 4: Transcripts down-regulated in cocaine overdose victims.
Figure imgf000039_0001
Figure imgf000040_0001
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Figure imgf000047_0001
Table 5: Transcripts up-regulated in cocaine overdose victims.
Figure imgf000047_0002
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Figure imgf000068_0001
Figure imgf000069_0001
Functional clustering was used to assess differential gene expression in biochemical pathways (e.g., cAMP and phosphoinositol pathways), protein families (e.g. dopamine receptors); protein motifs (e.g., zinc finger, leucine zipper, etc.) and transcripts previously identified as associated with cocaine abuse in human and animal models.
Reverse Northern Analysis was used as the secondary screen for 192 transcripts that were identified as differentially expressed (> 1.8 fold) in the high density microarray or as candidate genes that did not appear on the microarray. The resulting data are consistent with neuroadaptive response in intracellular signaling cascades associated with long-term cocaine-induced stimulation of dopamine receptors. Therefore, expression levels of a number of dopamine and signal transduction levels were determined by reverse Northern analysis to deteπriine the pattern of gene expression and indicate whether the VTA in cocaine overdose victims is preferentially affected as a consequence of long-term cocaine use. mRNA from each subject for each group was hybridized separately to two reverse Northern blots each containing 96 candidate clones. PCR was performed to amplify the inserts of the clones of interest. Approximately 250 ng of PCR material from each clone was spotted on Hybond XL nylon membrane. Probes were generated as described for Example 1 and hybridized to reverse Northern blots. Blots were exposed to phospho screens and desitometric analysis was performed using ImageQuant software.
Secondary screening demonstrated significant alterations in the expression of multiple genes and of several function-related gene groups. Chronic cocaine exposure enhances DA neurotransmission, which may result in regulatory changes in postynaptic DA and D2 receptors. Dl and D2 receptors are coupled to G s and Gαi proteins, respectively, and function to stimulate and inhibit AC activity. The data demonstrate an increase in G s and Gαi mRNA in the VTA of cocaine overdose victims, as well as significant differences in γ2 and γ5 subunits. RGS proteins regulate G-protein signaling by increasing the intrinsic GTPase activity of Gαi, Gαo, Gαt, and Gαq as well as Gsα. RGS 1-4 and 10 showed greater than 2 fold differential expression in the VTA of cocaine overdose victims. Although the present invention is not bound by a particular mechanism, these data suggest that RGS proteins are located in dopamine pathways and maybe involved in neuroadaptations in signal transduction mechanisms associated with chronic cocaine exposure. Experimental Procedures
1. Target and Probe Preparation cDNA from the clones are amplified, purified, and aliquoted into 96-well plates using a Quiagen BioRobot 3000. Universal primer pairs are used for PCR. Clones are amplified, and an aliquot is electrophoresed on an agarose gel for quality control, and any failed reaction (e.g. a reaction resulting in no or multiple bands) is repeated. cDNA is purified from the PCR reactions and an aliquot is used for fluorometric quantification. Samples are deleted in 50% DMS) to prevent evaporation during spotting. Plates containing the products are loaded onto a Cartesian Microarray and spotted at a concentration of 10 ng per spot. Each clone is spotted three times in different areas of the array. The arrayer uses a quill type spotter and is enclosed in a temperature and humidity controlled chamber. Pre-spotting 10 times increases the reproducibility of the amount of product spotted on arrays. Microarrays are dried at 37°C for one hour, and are then irradiated by ultraviolet to cross-link the DNA. Microarrays are washed with 70% ethanol, dried at 37°C and stored in dessicators at 4°C until needed.
To produce probes, approximately 400 ng of RNA is incubated at 70°C for 20 minutes, then quick cooled in ice for 5 minutes. Oligo dT-24 (400 ng), 250 μM each of dATP, dGTP, dCTP, and Cy 3.5 or Cy 5.5 labeled dUTP (Amersham Pharmacia Biotech.), 10 mM DTT and reverse transcriptase buffer to a final concentration of IX are added to the mRNA. The sample is incubated at 37°C for 30 minutes. On μl of
SUPERSCRIPT ® brand RNase H- reverse transcriptase (invitrogen) is added to the solution and incubated at 37°C for 90 minutes.
2. Hybridization
Hybridization of microarrays is performed using a Genomic Solutions GenTac Hybridization Staion. Both labeled probes are added to the hybridization chamber and are agitated genetly at 48°C for 24 hours. Following hybridization, a medium stringency was buffer (2X SSC/0.1% SDS) is applied to the slides with a buffer flow time of 45 seconds at 40°C for 6 minutes. A second was step with a buffer of higher stringency (0.5X SSC/0.1% SDS) is applied to the slides with a buffer flow time of 45 seconds at 40°C for 3 minutes. 2X SSC is used as the post wash buffer with a flow time of 40 seconds at 25° for 30 seconds each. Slides are dried at 25°C for 5 minutes before image analysis. 3. Image Analysis
Following hybridzation, microarrays are scanned using the GSI Lumonics ScanArray 5000. Initial image analysis is performed using ScanArray software, and are subsequently analyzed using GLEAMS software (NuTec Services). The levels of the Cy3 and Cy5 signals are determined, and the size and spacing of the dots on the microarray are defined and corrected for artifacts. The background is calculated for each spot using the immediate area surrounding the target spot. Controls are used to adjust channel intensity and to calculate the overall expression mean of each gene. Criteria for inclusion of spots for analysis include: 1) >4-fold signal intensity over background, 2) a ' symetric spot (not intensity) patter for each channel 3) lack of significant artifactual signal intensity (intensity recognition pattern algorithm in software and visual identification, and 4) hybridization present in 2 of the three spots per gene.
4. Reverse Northern Analysis
In order to produce blots for reverse northern analysis, clones are amplified and analyzed on agarose gels as described above. 250 ng of each amplified insert is spotted on HyBond XL ® net nylon transfer membrane (Amersham Pharmacia Biotech) using a 96 well dot blot apparatus (Schleicher and Schuell). DNA is crosslinked to the membrane by ultraviolet radiation. This amount of cDNA ensures that the cDNA is in vast excess to the respective aRNA, so that the target will not limit hybridization. Arrays will be hybridized for 48 hours at 44°C in a rotisserie hybridization oven with the following hybridization solution: 50% formamide, 5X SSC, 5X Denhard's solution, 0.1% SDS, 200 ng of sheared salmon sperm, and 1.0 mM sodim pyrophosphate. Following hybridization, slots blots are washed sequentially with 2X SSC/0.1% SDS, 0.5X SSC/0.1% SDS, and 0.1X SSC/0.1% SDS for 20 minutes each at 44°C. Labeled hybridized products are detected using phosphorimager cassettes, Image StormScanner and ImageQuant software (Molecular Dynamics).
5. Statistical Analysis
Expression levels from cocaine overdose victims and controls will be compared using univariate statistics for continuous variables and central tendency will be measured using the Wilcoxon signed rank sum test. Crude OR's and 95% confidence intervals will be estimated by the Mantel Haenszel method. 95% confidence intervals for all odds ratios are calculated with unconditional maximum likelihood. In order to determine the significance of differential gene expression between control and cocaine exposure groups, experimental data will be analyzed by a regression analysis.
The specific signal (corrected for background) of probe bound to each clone is expressed as a ratio of the total hybridization intensity of the array, thereby minimizing variations due to differences in the specific activity of the probe and the absolute quantity of probe present. Differential expression greater than 1.8-fold is accepted as above background and relevant for further examination. Relative changes in individual mRNAs for the respective brain regions is analyzed using a two-way ANOVA (analysis of variance between groups) with Group (cocaine overdose, controls) and Region (VTA vs. 1-SN; NAc vs. d-CP) as the fixed effects. The null hypothesis is rejected when P<0.05. Post hoc analyses are conducted as needed using Fisher least significance difference. Differentially expressed genes are grouped (clustered) into functional categories (e.g. signal transduction pathways, protein families such as receptors, RGS proteins, G-protein subunits, etc.) as well as to provide information on coordinate gene expression in a functional context. Differential gene expression is examined for relationships with indices of cocaine use (e.g. blood and brain concentrations; frequency and duration of use) by one-way ANOVA followed by post hoc analyses where appropriate. mRNA abundance serves as the dependent measure. Additional clinical data, including age, postmortem interval, sex, and race are examined for effects on gene expressing using ANOVA or correlation analysis, as appropriate. To confirm that the cocaine users and comparison subjects are well matched, age, sex, race, socioeconomic status, and postmortem interval will be compared by using two-tailed t tests. The parameters of the appropriate regression function (i.e., linear or quasilinear) will be estimated and the correlation coefficient determined. Comparisons between means will be made using least significant difference with PO.05. Similar analysis is performed by hemispheric laterality. Example 3: Alterations in Ionotropic Glutamate Receptor Subunits during Binge Cocaine Self- Administration and Withdrawal in Rats
I. Introduction Ionotropic glutamate receptors are classified as NMD A (NRl, NR2A-D,
NR3), (±)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMP A; GluRl - 4), and kainate (GluR5-7, KA1-2) receptor subunits based on their pharmacological characteristics and sequence information (Hollmann and Heinemann, S. (1994) Annu. Rev. Neurosci. 17, 31-108. Borgesmand Dingledine (2002) Molecular pharmacology and physiology of glutamate receptors, in Glutamate and addiction, Herman et al, eds, pp 3-22. Humana Press, Totawa, NI). Whereas AMPA and kainate subunits contribute to fast neurotransmission, all three ionotropic subtypes are thought to play roles in long term potentiation. Since subunit composition determines the functional properties of ionotropic glutamate receptors, alterations in ionotropic glutamate receptors in the VTA may indicate alterations in the excitability of dopamine transmission underlying long term biochemical and behavioral effects of cocaine which, in turn, may affect subsequent drug intake.
The effect of cocaine self-administration on alterations in the abundance of glutamate receptor subunits has not been described in the art. The present study was undertaken to evaluate changes in the abundance of iGluR subunit expression in mesocorticolimbic structures involved in cocaine reinforcement (VTA, NAc, PFC) following binge cocaine self-administration and withdrawal from cocaine. Similar changes were evaluated in the nigrostriatal pathway (SN and dorsal striatum) as a measure of regional and pathway specificity.
II. Materials and Methods A. Subjects
Male Sprague-Dawley rats (60-90 days; 225-275g; Charles River, Wilmington, MA) were housed individually in operant conditioning cages in a temperature-controlled vivarium on a 12 hr reversed light-dark cycle (lights on: 8:00 p.m.) with food and water available ad libitum throughout the experiment. B. Intravenous catheterization
Rats were anesthetized with halothane and implanted with chronic indwelling venous catheters, as described previously (Hemby et al. (1999) J Pharmacol. Exp. Ther. 288:274-80; Hemby et al. (1995) J Pharmacol. Exp. Ther. 273:591-98; Hemby (1997b) Psychopharmacology ( eά). 133:7-16). Catheters were inserted into the right jugular vein, terminating just outside the right atrium and anchored to muscle near the point of entry into the vein. The distal end of the catheter was guided subcutaneously to exit above the scapulae through a Teflon shoulder harness. The harness provided a point of attachment for a spring leash connected to a single channel swivel at the opposing end. The catheter was threaded through the leash for attachment to the swivel where the fixed end of the swivel was connected to a syringe by polyethylene tubing. Infusions were administered by a motor driven syringe pump controlled by a computer. Infusions of methohexital (100 μl; 10 mg/kg; i.v.) were administered to assess catheter patency, as needed. Health of the rats was monitored thrice daily by the experimenter and biweekly by institutional veterinarians according to the guidelines issued by the Emory University histitutional Animal Care and Use Committee and the National institute of Health.
C. Self-administration Procedures Subjects were housed in standard operant conditioning chambers
(24.5x23.5x2 lcm) containing a retractable lever and a stimulus light mounted directly above the lever. The chambers were enclosed in sound-attenuating boxes containing an exhaust fan, a house light, a tone source, and a water bottle. A motor driven syringe pump was located on the side of this external chamber. Extraneous noise was masked by the exhaust fan. Immediately following surgery, rats were placed in their respective chambers where they received infusions of heparinized 0.9% bacteriostatic saline (1.7 U/ml; 200 μl/30min) for 48 hr. On the following day, the self- administration procedure was initiated.
Rats were randomly divided into two groups: (BINGE access and WITHDRAWAL) and allowed to self-administer cocaine (0.5 mg/infusion; 200 μl/infusion; 6.2 sec/infusion) during an 8 hr. self-administration sessions (dark phase of the light cycle) under a fixed ratio-5 (FR5); time out 20 seconds schedule of reinforcement. Upon completion of the response requirement, a cocaine infusion was delivered and a 20-sec time-out was in effect. Responding was initially maintained under an FR1 that was gradually increased to FR5. During the time-out, the lever light was extinguished, the house light illuminated, and a tone was generated. The end of the time-out was signaled by illumination of the lever light and the house light and tone were extinguished. During the time-out, lever responses were recorded but had no scheduled consequence. IBM compatible computers were used for session programming and data collection. Once attaining the terminal ratio of FR5, rats were given limited access (8hrs/day, 7days/week) for 14 consecutive days. On the 15th day, the self-administration session was changed to multiple 3 hr access components separated by one hr time outs (TO) as depicted below:
Figure imgf000076_0001
Twenty-four sessions were looped such that the program began each day at the beginning of the dark cycle. After completion of the self-administration session on the 20th day, rats in the BINGE access group were sacrificed. On days 21-34, rats in the WITHDRAWAL group remained in the self-administration chambers but did not have access to cocaine or related stimuli and were sacrificed on day 35.
D. Tissue preparation and Western blot analysis For sacrifice, rats were anesthetized with halothane and intracardially perfused with phosphate buffered saline (pH=7.2). Brains were removed and sectioned on ice in the coronal plane using a brain matrix. Areas of interest were dissected immediately on ice-cooled aluminum plates from 1-mm slices (approximately +3.2 to +2.2, PFC; +1.7 to 0.7, NAc; 0.48 to -0.4, striatum; -5.2 to -6.2, SN/VTA; all measures relative to bregma (Paxinos and Watson (1998) The rat brain in stereotaxic coordinates, 4th edition, Academic Press); and immediately frozen at -80°C in Eppendorf tubes. The sample size for the Binge, Withdrawal and Control groups for the NAc, PFC and striatum was n=8 per region. Due to the size of the VTA and SN, two samples were pooled such that n=4 for the Withdrawal, Binge and control groups for these regions. Tissue samples were homogenized in 10 mM HEPES, 10 mM NaCl, 1 mM KH2PO4, 5 mM NaHCO3, ImM CaCl2, 0.5 mM MgCl2, 5 mM EDTA and the following protease inhibitors (PI): ImM phenylmethylsulfonylfluoride, 10 mM benzamidine, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 μg/ml pepstatin and centrifuged using a Beckman Coulter SW55Ti swinging bucket rotor at 5333 g for 5 min. Supernatant (cytosol and crude membrane) was removed and centrifuged at 59,255 g for 30 min at 4°C and the pure cytosolic supernatant was removed and stored at -80°C. The pellet containing the crude plasma membrane was re-suspended in 20 mM Tris HCl, 1 mM EDTA (pH=8.0) and 300 mM sucrose with Pis and centrifuged at 5333 g for 5 min. This procedure was repeated twice and the pellet was resuspended in phosphate buffered saline and stored at -80°C (crude plasma membrane fraction). The pellet from the intial centrifugation was re-suspended in 10 mM Tris (pH=7.5), 300 mM sucrose, 1 mM EDTA (ρH=8.0), 0.1% NP40 and Pis and centrifuged at 2370 g for 5 min at 4°C. The supernatant was discarded and the pellet was re-suspended in the buffer and washed three times before re-suspension in the PI buffer and storage of samples at -80°C (nuclear fraction), as described previously in Tang et al. (2003) J. Neurochem. 85:911 -24.
Protein concentrations were calculated using the bicinochoninic acid protein assay kit (Pierce, Rockford, IL) and diluted in Laemmli sample buffer to achieve the equivalent final protein concentrations. Five micrograms of protem were loaded into 10% sodium dodecyl sulfate-polyacrylamide gel, electrophoresed and transferred to nitrocellulose by electroblotting (30V, overnight at 4°C) in IX transfer buffer (Bio- Rad, Richmond, CA). Nitrocellulose membranes were blocked in 0.5% w/v nonfat dry milk and 0.1% v/v Tween 20 in phosphate-buffered saline (pH 7.4, 0.12 M) for 1 hr at room temperature prior to being incubated with primary antibodies in blocking buffer (Bio-Rad) overnight at 4°C followed by secondary antibody for one hr at room temperature. Protein bands were visualized on a Kodak XAR-5 film with enhanced chemiluminescence (ECL plus, Amersham Pharmacia Biotech). Primary antibodies were as follows: mouse monoclonal antibodies directed against NMDARI (Chemicon International, Temecula, CA) and rabbit polyclonal antibodies directed against NMDAR2A, NMDAR2B, NMDAR3A, NMDAR3B, GluRl, GluR2/3, GluR4, GluR5, GluR6/7, and KA2 (Upstate Biotechnology Cell Signaling Systems). Equal protein loading was confirmed by stripping the blots and re-probing them with a monoclonal /3-tubulin antibody (Upstate Biotechnology Cell Signaling Systems, Waltharn, MA; 1 :5000 v/v) followed by incubation with secondary antibody and visualization as described above. No significant differences were detected in β- rubulin abundance between the groups for any of the blots indicating that any differences in ionotropic glutamate receptor abundance between the groups was not due in unequal loading of protein in the gels. Protein abundances were calculated by optical densitometry with a Scan Jet 2200C and imported into NTH Image
1.61 software for analysis. Film background was subtracted from the optical density values to give the optical density value for each subject. All assays were conducted under conditions in which densitometric signal intensity was linear with protein concentration as determined by preliminary experiments. Data were expressed as percent of control levels (mean ± S.E.M.).
E. Data Analysis
Self-administration data were analyzed by two-way ANOVA with repeated measures (Time) with number of infusions as the dependent measure. Data for each iGluR subunit/region were normalized based on control levels and were analyzed using one way ANOVA. Null hypotheses were rejected when PO.05.
III. Results A. Behavioral data. Cocaine engendered and maintained rates of self-administration. There was no statistically significant difference in the number of infusions, and likewise cocaine intake, between the two groups over the course of the experiment [F(l, 377)=0.401, P=0.544]. T he total number of infusions was 895.8 (± 101.7) for the BINGE group and 981.3 (± 232.9) for the WITHDRAWAL group. During the 15 days of limited access, rats in the BINGE group averaged 551.3 (± 60.9) infusions/275.7 (± 30.5) mg of cocaine; whereas the WITHDRAWAL group averaged 621.7 (± 42.7) infusions/310.9 (± 21.4) mg of cocaine. Similarly, during the six days of unlimited access, the BINGE group self-administered approximately 344.4 (± 48.3) infusions/172.2 (± 24.2) mg of cocaine and the WITHDRAWAL group self- administered 359.5 (± 48.0) infusions/179.8 (± 24.0) mg of cocaine.
B. Regional comparison of iGluR subunit protein levels in controls. Analysis of the relative abundance of iGluR subunits across various brain regions was performed. Western blots of the iGluR subunits in each of the brain regions studied revealed single bands at the appropriate molecular weight. For the NMDA subunits, NMDARI was most abundant in the hippocampus, followed by the PFC, the NAc and striatum (HIPP>PFC>NAc, striatum» VTA, SN). There were no apparent differences in abundances of NMDAR2A, 2B, or 3 A among the hippocampus, PFC, NAc and striatum while these subunits were in low abundance in the VTA and SN. Interestingly, the NMDAR3B subunit appeared to be most abundant in the PFC and striatum followed by the hippocampus and NAc. Due to the paucity of protein from the VTA and SN, NR3A and 3B levels were not assessed. The abundances of GluRl, GluR2/3 and GluR4 were greater in the hippocampus and PFC than the NAc and striatum (HIPP, PFC>NAc, striatum>VTA, SN). GluR5 protein levels appeared to be equally abundant in all regions tested. For the kainate receptor subunits, GluR6/7 was most abundant in the NAc, striatum and PFC followed by the hippocampus, then the SN and VTA. In contrast, KA2 was most abundant in the NAc, moderately abundant in the striatum, PFC and hippocampus and least abundant in the VTA and SN.
C. Effects of cocaine self-administration history on iGluR subunit levels NRl: Analysis of variance revealed a significant effect of cocaine history on protein levels in the SN [F(2,16)=9.785; P-0.002] and striatum [F(2,22)=l 1.217; PO.001]. No significant differences were observed in the VTA, NAc or PFC. Post hoc analyses revealed increased levels in the SN following binge access which returned to control levels following withdrawal, whereas levels in the striatum following binge access and withdrawal were both significantly greater than control levels. NR2B: There was a significant effect of cocaine history on NR2B levels in the striatum [F(2,22)=12.996; P .001] and PFC [F(2,18)=11.881; PO.001]. Similar to NRl, NR2B levels in the striatum were increased following binge access and withdrawal; both at levels significantly greater than control levels. In the PFC, levels were significantly greater following withdrawal than either control or binge access.
NR3A: Analysis of variance revealed a significant effect of cocaine history on protein levels in the striatum [F(2,22)=12.169; P .001] and PFC [F(3,26)=3.928; P=0.021]. Due to the relatively small amounts of available protein in the VTA and SN, NR3A levels were not assessed in these regions. In the striatum, binge access produced significantly greater levels compared to control and withdrawal. In the PFC, protein levels were significantly reduced following withdrawal below levels observed in controls and binge access.
NR3B: Protein levels were significantly altered by cocaine in the striatum
[F(2,23)=101.821; P .001]. No changes were observed in the other brain regions. As noted for NR3 A, levels were not assessed in the VTA or SN. Levels following binge access were significantly greater than withdrawal which, in turn, were greater than control levels.
GluRl: Analysis of variance revealed a significant effect of cocaine history on protein levels in the VTA [F(2,16)=5.924; P=0.014], SN [F(2,19)=8.890; P=0.002], striatum [F(2,22)=42.004; PO.001] and PFC [F(2,20)=4.431; P=0.027], while there was no significant difference in the NAc. In the VTA, GluRl levels were significantly greater during withdrawal compared with binge access. In contrast, GluRl levels in the SN were increased during binge access but returned to control levels during withdrawal. In the striatum, levels were significantly elevated during withdrawal only (Figure 4A).
GluR2/3: Protein levels were significantly altered in the VTA
[F(2,l l)=5.494; P=0.028], NAc [F(2,22)=5.105; P=0.016], striatum [F(2,22)=38.918; P .001] and prefrontal cortex [F(2,20)=6.953; P=0.006], although no significant differences were observed in the SN. In the VTA, NAc, and PFC, withdrawal produced significantly greater levels than binge access. In the striatum and PFC, GluR2/3 levels following withdrawal were significantly more abundant than control levels.
GluR4: ANOVA revealed significant effects of cocaine on GluR4 protein levels in the striatum [F(2,33)=23.629; P=0.007] and PFC [F(2,19)=8.211; P=0.003]. There was no significant difference in the VTA, SN or NAc. Similar patterns of changes were observed in the striatum and PFC with levels following withdrawal significantly increased above both control and binge levels.
GluR5: There was a significant effect of cocaine history on GluR5 levels in the VTA [F(2,ll)=5.583; P=0.027], SN [F(2,16)-4.032; PO.041] and striatum [F(2,16)=l 0.706; P=0.002]. No significant differences between the groups were observed in the NAc or PFC. In the VTA, levels were significantly decreased during binge access compared with control levels, hi contrast, GluR5 levels in the SN increased during binge cocaine access and returned to control levels during withdrawal, h the PFC, levels were significantly increased during withdrawal above both control and binge levels.
GluR6/7: ANOVA revealed significant effects of cocaine on GluR6/7 protein levels in the VTA [F(2,l l)= 20.731; PO.001], SN [F(2,20)=4.232; PO.031], and striatum [F(2,22)=23.934; PO.001]. No significant differences were observed in the PFC or NAc, although there was a trend towards significance in the NAc [F(2,22)=:3.339; P=0.056]. h the VTA, protein levels were significantly decreased during binge access but returned to control levels following two weeks of withdrawal. In contrast, SN GluR6/7 levels increased following binge cocaine access and returned to control levels during withdrawal. In the striatum, GluR6/7 protein levels were increased above control and binge access levels following two weeks of withdrawal (Figure 6B).
KA2: ANOVA revealed significant effects of cocaine on protein levels in the SN [F(2,20)=l 1.357; PO.001] and striatum [F(2,22)=42.004; PO.001]. There were no significant differences in KA2 levels between the groups in the VTA, NAc or PFC. Levels were significantly increased in the SN during binge access and returned to control levels following withdrawal, hi contrast, KA2 protein levels were significantly increased during withdrawal but were not different from control levels following binge access (Figure 6C).
Figure imgf000082_0001
IV. Discussion In the present study, Western blot analysis was used to examine the expression of iGluR protein subunits following binge cocaine self-administration and two weeks of withdrawal in brain the mesocorticolimbic and nigrostriatal dopamine pathways. The study demonstrated that binge cocaine self-administration and withdrawal induce changes in protein levels of iGluR subunits in a region specific manner and were dependent upon the history of cocaine exposure. The present results provide the first composite assessment of iGluR subunit protein alterations in mesocorticolimbic brain regions associated with cocaine reinforcement (VTA, NAc, and PFC) compared with regions in the nigrostriatal pathway (SN and striatum).
A. NMDA receptor subunits
In the present study, no significant differences were observed in the VTA, NAc or PFC NRl levels or following binge cocaine self-administration. However, NRl protein levels were increased following six days of binge cocaine access in the SN compared to control and withdrawal levels and in the striatum following binge and withdrawal compared to control levels. Interestingly, increased levels returned to baseline following 2 weeks of withdrawal in the SN whereas levels remained elevated following withdrawal in the striatum. In the present study, NR2B and NR3B levels were increased in the striatum following binge cocaine self-administration and remained elevated above control levels following two weeks of withdrawal suggesting these changes may represent long-term alterations in NMDA receptor function following cocaine exposure, hi contrast, NR3 A levels in the striatum were increased following binge cocaine access but returned to control levels following two weeks of withdrawal. In the PFC, withdrawal from cocaine increased NR2B above control and binge access levels whereas withdrawal levels of NR3A were decreased compared to control and binge access levels. The presence of NR3A subunits, which must co-express with NRl for
• 94- membrane expression, leads to decreased Ca permeability through the NMDA receptor complex. Since increased Ca2+ permeability is necessary for long-term synaptic changes, the present data suggest an increased opportunity for LTP to occur in mPFC neurons. From a functional perspective, increased NR2B levels indicate a slow deactivation time (-400 msec) for the receptor complex, which when paired with decreased expression of NR3A may yield an increase in Ca2+ permeability and hyperexcitability of prefrontal function. NRl subunits are required for the normal function of the NMDA ionophore. The subunit is phosphorylated by PKA, PKC and possibly by CamKII which cause increased Ca2+ influx tlirough the activated receptor leading to the slow onset of the EPSP. NRl subunits may lead to enhanced Ca2+ influx in dopamine neurons resulting in hyperexcitability of these cells. With no apparent change in NRl protein levels, NR3A alterations may provide an alternative means to increase intracellular Ca2+ levels without altering the number of receptors.
B. AMPA receptor subunits GluRl and GluR2/3 levels in the VTA were significantly increased during withdrawal compared with binge access. Similarly in the NAc, GluR2/3 levels were increased following withdrawal from cocaine compared with binge access, an effect also observed in human cocaine overdose victims. Significant upregulation of GluR2/3 and GluR4 in the PFC were observed following withdrawal compared with control and binge access levels.
In the nigrostriatal pathway, GluRl levels in the SN were increased during binge access compared to control and withdrawal levels whereas AMPA subunit levels in the striatum were increased during two weeks of withdrawal from cocaine compared to control and binge access levels. In addition, binge GluR2 levels in the striatum were significantly lower than control and withdrawal levels. Previous studies using experimenter-administered cocaine, did not observe significant alterations in AMPA subunits in the nigrostriatal pathway following chronic cocaine or withdrawal (Fitzgerald et al. (1996) J. Neurosci. 16,:274-82; Churchill et al. (1999). J.
Neurochem. 72:2397-403), again suggesting the potential relative importance of contingent drug administration.
C. Kainate receptor subunits GluR6 and KA2 exhibited similar moderate to strong immunoreactivity in the
NAc, striatum and PFC and light immunoreactivity in the SN and VTA. GluR5 levels were moderate to strong in the PFC and were similarly expressed in the NAc, striatum, VTA and SN. Interestingly, GluR5 levels were expressed at considerably higher levels in the rat compared with human VTA. Unlike NMDA and AMPA receptor subunits, the physiological function of kainate receptors remains unclear; however, they appear to be important for controlling Ca2+ influx through the kainate ionophore in different pathological states, such as cocaine addiction (Paschen and Djuricic 1994).
In the present study, GluR5, GluR6/7 and KA2 kainate receptor subunit levels were regulated in a region specific manner. In the VTA, GluR5 and GluR6/7 immunoreactivities were decreased during binge access, whereas binge access increased GluR5, GluR6/7 and KA2 in the SN compared with withdrawal levels. Conversely, the kainate receptor subunits in the striatum were significantly upregulated during withdrawal compared with control and binge access levels. Presently, there is a paucity of information on the regulation and function of kainate receptor subunits and even less about the role of these subunits in cocaine abuse and addiction.
V. Summary The present study demonstrated the regional and subunit-specific changes in iGluR protein expression following binge cocaine self-administration and withdrawal. Theseresults provide a significant addition to the knowledge of altered glutamatergic function induced by chronic cocaine self-administration and withdrawal. The study is unique in that it is the first study to assess alterations of multiple iGluR subtypes in various brain regions following binge cocaine self-admimstration and withdrawal. Such changes may be related to behaviors associated with withdrawal such as decreased locomotion, increased anxiety and behavioral sensitization and other enduring effects and may be an important mechanism by which cocaine exerts long- term effects on the mesolimbic dopamine system.
All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

THAT WHICH IS CLAIMED:
1. A method for identifying genes and their expression products as screening targets for drugs for treating addictive disorders, the method comprising: (a) determining the expression level of one or more genes in a first sample from one or more subjects affected by an addictive disorder;
(b) determining the expression level of said one or more genes in a second sample from one or more control subjects;
(c) for each of said one or more genes, comparing the expression level measured in step (a) with the expression level measured in step (b); wherein a gene whose expression level is significantly increased or significantly decreased in said one or more subjects affected by an addictive disorder in comparison with the expression level in said one or more control subjects is identified, along with its expression products, as a screening target for drugs for treating addictive disorders.
2. The method of claim 1, wherein said first sample and said second sample comprise cells that are affected by addictive disorders.
3. The method of claim 2, wherein said cells that are affected by addictive disorders are selected from the group consisting of ventral tegmental area cells and lateral substantia nigra cells.
4. The method of claim 1 wherein said addictive disorder is cocaine addiction.
5. The method of claim 1, wherein said first sample and said second sample comprise cells from human post-mortem tissues.
6. A method for producing an expression profile comprising values representing the expression levels of genes whose expression is associated with addictive disorders, the method comprising: (a) determining the expression level of one or more genes in a first sample from one or more subjects affected by an addictive disorder;
(b) determining the expression level of said one or more genes in a second sample from one or more control subjects; (c) identifying genes whose expression level is significantly increased or significantly decreased in said one or more subjects affected by drug addiction in comparison with the expression level in said one or more control subjects; and
(d) producing an expression profile comprising values representing the expression levels of genes of genes identified in step (d) in subjects affected by an addictive disorder.
7. The method of claim 6, wherein said one or more genes are selected from the group consisting of the genes shown in Tables 3, 4, and 5.
8. The method of claim 6, wherein said expression profile comprises values representing the expression levels of at least five genes
9. The method of claim 6, wherein said expression profile comprises values representing the expression levels of at least ten genes
10. The method of claim 6, wherein said expression profile comprises values representing the expression levels of at least ten genes selected from the group consisting of the genes shown in Tables 3, 4, and 5. (
11. The method of claim 6, wherein said expression profile comprises values representing the expression levels of genes selected from the genes shown in Table 3.
12. The method of claim 6, wherein said first sample and said second sample comprise cells selected from the group consisting of ventral tegmental area cells and lateral substantia nigra cells.
13. The method of claim 12, wherein said first sample and said second sample comprise at least 50% of a cell type selected from the group consisting of ventral tegmental area cells and lateral substantia nigra cells.
14. The method of claim 6 wherein said addictive disorder is cocaine
, addiction.
15. The method of claim 6 wherein said first sample and said second sample comprise cells from human post-mortem tissues.
16. A method for predicting whether a compound will be addictive in humans, said method comprising:
(a) providing a reference expression profile associated with an addictive disorder, wherein said expression profile comprises values representing the expression level of one or more genes whose expression is associated with an addictive disorder in one or more samples from one or more subjects affected by the addictive disorder;
(b) providing a test expression profile comprising values representing the expression level of said one or more genes in a sample comprising cells that been contacted with the compound; and
(c) determining whether said test expression profile shares sufficient similarity to said reference profile; wherein a determination that the test expression profile shares sufficient similarity to the reference profile results in a prediction that the compound will be addictive in humans.
17. The method of claim 16, wherein said reference expression profile is produced according to the methods of claim 6.
18. The method of claim 16, wherein said reference expression profile is produced according to the methods of claim 7.
19. The method of claim 16, wherein said reference expression profile comprises values representing the expression levels of at least five genes selected from the group consisting of the genes shown in Tables 3, 4, and 5.
20. The method of claim 16, wherein said reference expression profile comprises values representing the expression levels of genes selected from the genes shown in Table 3.
21. In a method of screening for therapeutic compounds for use in treating addictive disorders, an improvement comprising the use of a target identified by the method of any one of claims 1-4.
22. The method of claim 21, where wherein said addictive disorder is cocaine addiction.
23. A method of screening for therapeutic compounds for use in treating addictive disorders comprising screening for modulators of a target gene selected from the group consisting of the genes listed in Table 3, the genes listed in Table 4, the genes listed in Table 5, and the expression products of said genes.
24. A method of screening for therapeutic compounds for use in treating addictive disorders comprising screening for inhibitors of a target gene selected from the group consisting of the genes listed in Table 5 and the expression products of said genes.
25. A method of screening for therapeutic compounds for use in treating addictive disorders comprising screening for activators of a target gene selected from the group consisting of the genes listed in Table 4 and the expression products of said genes.
26. A method for evaluating a candidate drug to determine whether it will have therapeutic efficacy in treating an addictive disorder, the method comprising: (a) determining the expression level of one or more genes in a first sample from a subject affected by an addictive disorder prior to treatment with the candidate drug, wherein expression of said one or more genes is associated with the addictive disorder; (b) determining the expression level of said one or more genes in a second sample from the subject following treatment with the candidate drug;
(c) for each of said one or more genes, comparing the expression level measured in step (a) with the expression level measured in step (b) to determine the change in the expression level of said genes following treatment with the candidate drug.
27. The method of claim 26, wherein said one or more genes comprise at least five genes selected from the group consisting of genes shown in Tables 3, 4, and 5.
28. The method of claim 26, wherein said one or more genes are selected from the genes shown in Table 3.
29. The method of claim 26, where wherein said addictive disorder is cocaine addiction.
30. An array comprising a substrate having a plurality of addresses, wherein each address has disposed thereon a capture probe that can specifically bind a nucleic acid molecule that is differentially expressed in subjects affected by cocaine addiction.
31. The array of claim 30, wherein the nucleic acid molecule that is differentially expressed in subjects affected by cocaine addiction is selected from the group consisting of the genes shown in Tables 3, 4, and 5.
32. The array of claim 31 , wherein the substrate has greater than 10 addresses.
33. The array of claim 32, wherein the substrate has greater than 20 addresses.
34. The array of claim 33, wherein the substrate has greater than 50 addresses.
35. The array of claim 34, wherein the substrate has greater than 100 addresses.
36. The array of claim 31, wherein the substrate has no more than 500 addresses.
37. A kit for predicting whether a compound will be addictive in humans, the kit comprising (a) an array comprising a substrate having a plurality of addresses, wherein each address has disposed thereon a capture probe that can specifically bind a nucleic acid molecule that is differentially expressed in subjects affected by an addictive disorder; and
(b) a computer-readable medium having a digitally-encoded expression profiles having values representing the expression of a nucleic acid molecules detected by the array.
38. The kit of claim 37, wherein said array is the array of claim 31.
39. A computer-readable medium having a digitally-encoded expression profile comprising one or more values representing the expression levels of a gene that is differentially expressed in cocaine overdose victims.
40. The computer readable medium of claim 39, wherein the expression profiles comprise values selected from the group consisting of values representing the expression levels of the genes shown in Tables 3, 4, and 5.
41. The computer readable medium of claim 40, wherein the expression profiles comprise values selected from values representing the expression levels of at least 10 genes selected from the genes show in Tables 3, 4, and 5.
42. The computer readable medium of claim 41 , wherein the expression profiles comprise values selected from values representing the expression levels of at least 20 genes selected from the genes show in Tables 3, 4, and 5.
43. The computer readable medium of claim 42, wherein the expression profiles comprise values selected from values representing the expression levels of at least 50 genes selected from the genes show in Tables 3, 4, and 5.
44. The computer readable medium of claim 43, wherein the expression profiles comprise values selected from values representing the expression levels of at least 100 genes selected from the genes show in Tables 3, 4, and 5.
45. A kit use in a method of evaluating the therapeutic efficacy of a candidate drug in treating an addictive disorder, the kit comprising
(a) an array comprising a substrate having a plurality of addresses, wherein each address has disposed thereon a capture probe that can specifically bind a nucleic acid molecule that is differentially expressed in subjects affected by an addictive disorder; and
(b) a computer-readable medium having a digitally-encoded expression profiles having values representing the expression of a nucleic acid molecules detected by the array.
46. The kit of claim 45, wherein said array is the array of claim 31.
47. The method of claim 1, wherein said first sample and said second sample comprise cells from a rodent model for cocaine addiction.
48. The method of claim 6, wherein said one or more genes are selected from the group consisting of the genes shown in Table 3, 4, 5, and 6.
49. The method of claim 16, wherein said reference expression profile comprises values representing the expression levels of at least five genes selected from the group consisting of the genes shown in Tables 3, 4, 5, and 6.
50. A method of screening for therapeutic compounds for use in treating addictive disorders comprising screening for modulators of a target gene selected from the group consisting of the genes listed in Table 6, and the expression products of said genes.
51. The method of claim 26, wherein said one or more genes comprise at least five genes selected from the group consisting of genes shown in Tables 3, 4, 5, and 6.
52 The array of claim 30, wherein the nucleic acid molecule that is differentially expressed in subjects affected by cocaine addiction is selected from the group consisting of the genes shown in Tables 3, 4, 5, and 6.
53. The computer readable medium of claim 39, wherein the expression profiles comprise values selected from the group consisting of values representing the expression levels of the genes shown in Tables 3, 4, 5, and 6.
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