EP1331930A2 - Procedes et compositions pour reguler la consolidation de la memoire - Google Patents

Procedes et compositions pour reguler la consolidation de la memoire

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Publication number
EP1331930A2
EP1331930A2 EP01924598A EP01924598A EP1331930A2 EP 1331930 A2 EP1331930 A2 EP 1331930A2 EP 01924598 A EP01924598 A EP 01924598A EP 01924598 A EP01924598 A EP 01924598A EP 1331930 A2 EP1331930 A2 EP 1331930A2
Authority
EP
European Patent Office
Prior art keywords
gene
ltm
expression
memory
protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01924598A
Other languages
German (de)
English (en)
Other versions
EP1331930A4 (fr
Inventor
Cristina M. Alberini
Mark F. Bear
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Brown University Research Foundation Inc
Howard Hughes Medical Institute
Original Assignee
Brown University Research Foundation Inc
Howard Hughes Medical Institute
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Filing date
Publication date
Application filed by Brown University Research Foundation Inc, Howard Hughes Medical Institute filed Critical Brown University Research Foundation Inc
Publication of EP1331930A4 publication Critical patent/EP1331930A4/fr
Publication of EP1331930A2 publication Critical patent/EP1331930A2/fr
Withdrawn legal-status Critical Current

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity
    • G01N2333/4706—Regulators; Modulating activity stimulating, promoting or activating activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/65—Insulin-like growth factors (Somatomedins), e.g. IGF-1, IGF-2
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70571—Assays involving receptors, cell surface antigens or cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the initial phase of memory consolidation occurs in the first few minutes after we are exposed to a new idea or learning experience. If a learning experience has on-going meaning to us, the next week or so serves as a further period of memory consolidation. In effect, in this phase, the material moves from short-term memory to long-term memory for storage.
  • LTM long-term memory
  • introduction of a memory item alters the pattern of existing neuronal connectivity to form a neuronal network that will subserve the information for long-term storage.
  • Modulation of synaptic efficacy is induced by changes in synaptic transmission within selected synapses or alteration in synaptic contacts. These changes are in turn supported by molecules that underlie transmission or synaptic remodeling. It is suggested that modulation of gene expression is needed for LTM formation to overcome the relative short lifetime of proteins in neurons (as compared with enduring memory).
  • CREB is a nuclear protein that modulates the transcription of genes with cAMP responsive elements in their promoters. Increases in the concentration of either calcium or cAMP can trigger the phosphorylation and activation of CREB. Following its phosphorylation, CREB binds to the enhancer element CRE which is located in the upstream region of cAMP-responsive genes, thus triggering transcription. Some of the newly-synthesized proteins are additional transcription factors that ultimately give rise to the activation of late response genes, whose products are responsible for the modification of synaptic efficacy leading to LTM.
  • CREB subserves the formation of memories of various types of tasks that utilize different brain structures.
  • Evidence is available suggesting that CREB regulates the transcription of genes that subserve LTM.
  • CREB activation has been interfered with by microinjec ion of CRE containing oligonucleotides into cultured neurons.
  • CREB function has been disrupted using a reverse genetic approach.
  • LTM has been specifically blocked by the induced expression of a CREB repressor isoform, and enhanced by the induced expression of an activator isoform.
  • the role of CREB has been confirmed by behavioural analyses of a knock-out line with a targeted mutation in the CREB gene. In these mutants, learning and short term memory are normal, whereas long term memory is impaired. On the whole, the data suggest that encoding of long term memories involve highly conserved molecular mechanisms.
  • One aspect of the present invention provides a a method for modulating long term memory consolidation in an animal comprising treating an animal with an agent that modulates the activity of one or more of zif 268, insulin-like growth factor, glutamate receptor 1 (GluRl), glutamate receptor 2 (GluR2), c/EBP ⁇ and NGF.
  • Another aspect of the present invention relates to a method for enhancing long term memory consolidation in an animal comprising treating an animal with an agent that modulates a signal transduction pathway of glutamate receptor 1 (GluRl) or glutamate receptor 2 (GluR2), which agent is a ligand for the GluRl or GluR2 receptor.
  • Still another aspect of the present invention relates to a method for identifying an agent (e.g., as a drug discovery assay) which modulates memory consolidation, comprising,
  • reaction system for detecting the level of expression of a gene selected from the group consisting of of zif 268, insulin-like growth factor, glutamate receptor 1 (GluRl), glutamate receptor 2 (GluR2), c/EBP ⁇ and NGF; (ii) contacting said system with a test compound; and (iii) determining if the test compound alters the level of expression of the gene.
  • the reaction system is a cell-free system, such as a purified protein preparation or a cell-lysate. In other embodiments, the reaction system is a whole cell system.
  • the assay can be used to identify agents which modulate memory consolidation from amongst a plurality of different test agents.
  • the test compound can be small organic molecules, e.g., those having a molecular weight less than 2500 amu.
  • Still another aspect of the present invention provides a method of conducting a drug discovery business comprising:
  • the business method includes an additional step of establishing a distribution system for distributing the pharmaceutical preparation for sale, and may optionally include establishing a sales group for marketing the pharmaceutical preparation.
  • Yet another aspect of the present invention provides a method of conducting a target discovery business comprising:
  • Still another aspect of the invention provides a a pharmaceutical preparation comprising, one or more compounds identified in the above assays, formulated in a pharmaceutically acceptable excipient.
  • the preparation can also include one or more of a neuronal growth factor, a neuronal survival factor, and/or a neuronal tropic factor.
  • the subject pharmaceutical preparations can also include an agent that activates CREB-dependent transcription, such as a cAMP elevating agent, e.g., a cAMP agonist activates adenylate cyclase, cAMP analog, or a cAMP phosphodiesterase inhibitor.
  • a cAMP elevating agent e.g., a cAMP agonist activates adenylate cyclase, cAMP analog, or a cAMP phosphodiesterase inhibitor.
  • the subject pharmaceutical preparations can be administered to an animal as part of a treatment method for enhancing memory consolidation in an animal, or otherwise enhancing the functional performance of CNS neurons.
  • Yet another aspect of the invention provides a method for assessing a patient for learning and/or memory functional performance including a step of detecting the expression of, or a mutation in, one or more genes selected from the group consisting of zif 268, insulin-like growth factor, glutamate receptor 1 (GluRl), glutamate receptor 2 (GluR2), c/EBP ⁇ and NGF, or the level of activity of the gene products thereof, (optionally) in the patient's hippocampus.
  • Figure 1 Time-course Northern blot analysis of zif268 and c-fos following IA training. Increase in zif268, but not c-fos mRNA is evident in all animals at 9 and 20 hr after training.
  • Figure 2 Time course Northern blot analysis of C/EBP ⁇ and cyclophilin (control) mRNA following IA training in hippocampi of unoperated and fornix-lesion rats.
  • Figure 3 Broad scale expression profiling with cDNA expression arrays. Side by side hybridizations with cDNA probes prepared from two different RNA populations allow the simultaneous comparison of the expression levels of all the cDNAs on the array, (from Clontech user manual).
  • Figure 4 Examples of changes in gene array hybridizations reflecting differential expression of mRNAs following IA training. Hippocampi of control rats are compared to hippocampi of rats trained and sacrificed 9 hr later. Note that on these arrays each sequence is spotted in duplicate.
  • Figure 5 A Northern blot test to confirm the levels of certain transcripts.
  • Panel A Mean escape latency for rats trained on the water maze. Rats received eight trials a day, for four consecutive days.
  • Panel B Mean escape latency on each of the eight trials of day one (trials 1-8) and day two (trials 9-16).
  • the present invention is based on the discovery of genes that are up- or down-regulated in inhibitory avoidance, e.g., long-term memory, which genes are therefor believed to have roles in memory consolidation.
  • memory consolidation involves the regulation of expression of such genes as zif268 (EGRl), insulin-like growth factor (IGF-1), glutamate receptor 1 (GluRl), glutamate receptor 2 (GluR2), c/EBP ⁇ and VGF.
  • EGRl insulin-like growth factor
  • GluRl glutamate receptor 1
  • GluR2 glutamate receptor 2
  • test agents can be assessed in a cell-based or cell-free assay for ability to inhibit or potentiate the activity of an LTM protein, e.g., by modulating an enzymatic activity of the protein, modulating the half-life of the protein, modulating the interaction of the protein with other proteins, nucleic acids, carbohydrates or other biological molecules, modulating the cellular localization of the protein and the like.
  • Still another aspect of the invention relates to the use of compounds identified in the subject drug screening assays for altering (increasing or decreasing) the occurrence of learning and/or memory defects in an animal, and thus, altering the learning ability and/or memory capacity of the animal.
  • the compounds of the present invention may be useful as therapeutic agents in memory impairment, e.g., due to toxicant exposure, brain injury, epilepsy, mental retardation in children and senile dementia, including Alzheimer's disease.
  • Cells “host cells” or “recombinant host cells” are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • Complementary sequences as used herein refer to sequences which have sufficient complementarity to be able to hybridize, forming a stable duplex.
  • CREB cAMP response element
  • CREB is also phosphorylated via other kinases, such as described above and in Deisseroth et al. (1996) Neuron 16:89-101; Impey et al. (1996) Neuron 16:973-82; and Impey et al. (1998) Neuron 21:869-883.
  • genes for a particular polypeptide may exist in single or multiple copies within the genome of an individual. Such duplicate genes may be identical or may have certain modifications, including nucleotide substitutions, additions or deletions, which all still code for polypeptides having substantially the same activity.
  • the term "DNA sequence encoding" a polypeptide may thus refer to one or more genes within a particular individual.
  • certain differences in nucleotide sequences may exist between individual organisms, which are called alleles. Such allelic differences may or may not result in diffecrcnccs in amino acid sequence of the encoded polypeptide yet still encode a protein with the same biological activity.
  • the term “gene” or “recombinant gene” refers to a nucleic acid molecule comprising an open reading frame encoding a polypeptide, including both exon and (optionally) intron sequences.
  • the term “intron” refers to a DNA sequence present in a given gene which is not translated into protein and is generally found between exons.
  • Homology or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. The term “percent identical” refers to sequence identity between two amino acid sequences or between two nucleotide sequences. Identity can each be determined by comparing a position in each sequence which may be aligned for purposes of comparison.
  • the molecules are identical at that position; when the equivalent site occupied by the same or a similar amino acid residue (e.g., similar in steric and/or electronic nature), then the molecules can be referred to as homologous (similar) at that position.
  • homologous similar
  • Expression as a percentage of homology/similarity or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences.
  • Various alignment algorithms and/or programs may be used, including FASTA, BLAST or ENTREZ.
  • FASTA and BLAST are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default settings. ENTREZ is available through the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Md.
  • the percent identity of two sequences can be determined by the GCG program with a gap weight of 1, e.g., each amino acid gap is weighted as if it were a single amino acid or nucleotide mismatch between the two sequences.
  • interact as used herein is meant to include detectable interactions between molecules, such as can be detected using, for example, a two hybrid assay.
  • interact is also meant to include "binding" interactions between molecules. Interactions may be protein-protein or protein-nucleic acid in nature.
  • an isolated nucleic acid encoding a particular polypeptide preferably includes no more than 10 kilobases (kb) of nucleic acid sequence which naturally immediately flanks the gene in genomic DNA, more preferably no more than 5 kb of such naturally occurring flanking sequences, and most preferably less than 1.5 kb of such naturally occurring flanking sequence.
  • kb kilobases
  • isolated also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • isolated nucleic acid is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
  • isolated is also used herein to refer to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides.
  • modulation refers to both upregulation, i.e., stimulation, and downregulation, i.e. suppression, of a response.
  • non-human animals include mammalians such as rodents, non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc. Preferred non-human animals are selected from the rodent family including rat and mouse, most preferably mouse.
  • chimeric animal is used herein to refer to animals in which the recombinant gene is found, or in which the recombinant is expressed in some but not all cells of the animal.
  • tissue-specific chimeric animal indicates that one of the recombinant genes is present and/or expressed or disrupted in some tissues but not others.
  • nucleic acid refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides.
  • promoter means a DNA sequence that regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in cells.
  • tissue specific i.e. promoters, which effect expression of the selected DNA sequence only in specific cells (e.g. cells of a specific tissue).
  • leaky so-called “leaky” promoters, which regulate expression of a selected DNA primarily in one tissue, but cause expression in other tissues as well.
  • the term also encompasses non- tissue specific promoters and promoters that constitutively express or that are inducible (i.e. expression levels can be controlled).
  • protein protein
  • polypeptide peptide
  • recombinant protein refers to a polypeptide of the present invention which is produced by recombinant DNA techniques, wherein generally, DNA encoding a polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein.
  • phrase "derived from”, with respect to a recombinant gene is meant to include within the meaning of "recombinant protein” those proteins having an amino acid sequence of a native protein, or an amino acid sequence similar thereto which is generated by mutations including substitutions and deletions (including truncation) of a naturally occurring form of the protein.
  • Transcriptional regulatory sequence is a generic term used throughout the specification to refer to DNA sequences, such as initiation signals, enhancers, and promoters, which induce or control transcription of protein coding sequences with which they are operably linked.
  • transcription of one of the recombinant genes is under the control of a promoter sequence (or other transcriptional regulatory sequence) which controls the expression of the recombinant gene in a cell- type in which expression is intended.
  • the recombinant gene can be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences which control transcription of the naturally- occurring forms of proteins.
  • the term “transfection” means the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell by nucleic acid-mediated gene transfer.
  • "Transformation" refers to a piocess in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA or RNA, and, for example, the transformed cell expresses a recombinant form of a polypeptide or, in the case of anti-sense expression from the transferred gene, the expression of a naturally-occurring form of the protein is disrupted.
  • transgene means a nucleic acid sequence encoding s polypeptide or an antisense transcript thereto, which is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout).
  • a transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, (e.g. as intron), that may be necessary for optimal expression of a selected nucleic acid.
  • a "transgenic animal” refers to any animal, preferably a non-human mammal, bird or an amphibian, in which one or more of the cells of the animal contain heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.
  • the nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
  • the term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.
  • transgenic animal In the typical transgenic animals described herein, the transgene causes cells to express a recombinant form of a proteins, e.g. either agonistic or antagonistic forms.
  • transgenic animals in which the recombinant gene is silent are also contemplated, as for example, the FLP or CRE recombinase dependent constructs described below.
  • transgenic animal also includes those recombinant animals in which gene disruption is caused by human intervention, including both recombination and antisense techniques.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • One type of preferred vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication.
  • Preferred vectors are those capable of autonomous replication and/expression of nucleic acids to which they are linked.
  • Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors”.
  • expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer generally to circular double stranded DNA loops which, in their vector form are not bound to the chromosome.
  • plasmid and "vector” are used interchangeably as the plasmid is the most commonly used form of vector.
  • vector is intended to include such other forts of expression vectors which serve equivalent functions and which become lcnown in the art subsequently hereto.
  • III. Exemplary Embodiments A. Nucleic Acids encoding LTM proteins
  • LTM protein involved in memory consolidation
  • equivalent is understood to include nucleotide sequences encoding functionally equivalent LTM polypeptides or functionally equivalent peptides having an activity of an LTM protein such as described herein.
  • Equivalent nucleotide sequences will include sequences that differ by one or more nucleotide substitution, addition or deletion, such as allelic variants.
  • Preferred nucleic acids are vertebrate LTM nucleic acids. Particularly preferred vertebrate LTM nucleic acids are mammalian. Regardless of species, particularly preferred LTM nucleic acids encode polypeptides that are at least 80% similar to an amino acid sequence of a vertebrate LTM protein. In one embodiment, the nucleic acid is a cDNA encoding a polypeptide having at least one bioactivity of the subject LTM polypeptide.
  • nucleic acids of the present invention encode an LTM polypeptide which is comprised of at least 2, 5, 10, 25, 50, 100, 150 or 200 amino acid residues.
  • preferred nucleic acid molecules for use as probes/primer or antisense molecules i.e. noncoding nucleic acid molecules
  • coding nucleic acid molecules can comprise about 300, 400, 500, 600, 700, 800, 900, 950, 975, 1000 base pairs.
  • Another aspect of the invention provides a nucleic acid which hybridizes under stringent conditions to a nucleic acid encoding a cloned LTM gene.
  • Appropriate stringency conditions which promote DNA hybridization for example, 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 x SSC at 50°C, are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • the salt concentration in the wash step can be selected from a low stringency of about 2.0 x SSC at 50°C. to a high stringency of about 0.2 x SSC at 50°C.
  • the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22°C, to high stringency conditions at about 65°C. Both temperature and salt may be varied, or temperature of salt concentration may be held constant while the other variable is changed.
  • Preferred nucleic acids have a sequence at least 75% homologous and more preferably 80% and even more preferably at least 85% homologous with an nucleic acid sequence of an LTM gene. Nucleic acids at least 90%, more preferably 95%, and most preferably at least about 98-99% homologous with a nucleic sequence of an LTM gene are of course also within the scope of the invention.
  • Nucleic acids having a sequence that differs from the nucleotide sequences shown in one of SEQ ID NOs: 1-X due to degeneracy in the genetic code are also within the scope of the invention.
  • Such nucleic acids encode functionally equivalent peptides (i.e., a peptide having a biological activity of a LTM polypeptide) but differ in sequence from the sequence shown in the sequence listing due to degeneracy in the genetic code. For example, a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC each encode histidine) may result in "silent" mutations which do not affect the amino acid sequence of a LTM polypeptide.
  • LTM polypeptides which are isolated from, or otherwise substantially free of other cellular proteins, especially other signal transduction factors and/or transcription factors which may normally be associated with the LTM polypeptide.
  • the term "substantially free of other cellular proteins" (also referred to herein as "contaminating proteins") or “substantially pure or purified preparations” are defined as encompassing preparations of LTM polypeptides having less than about 20% (by dry weight) contaminating protein, and preferably having less than about 5% contaminating protein.
  • Functional forms of the subject polypeptides can be prepared, for the first time, as purified preparations by using a cloned gene as described herein.
  • purified it is meant, when referring to a peptide or DNA or RNA sequence, that the indicated molecule is present in the substantial absence of other biological macromolecules, such as other proteins.
  • the term “purified” as used herein preferably means at least 80% by dry weight, more preferably in the range of 95-99% by weight, and most preferably at least 99.8% by weight, of biological macromolecules of the same type present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 5000, can be present).
  • pure as used herein preferably has the same numerical limits as “purified” immediately above.
  • isolated and purified do not encompass either natural materials in their native state or natural materials that have been separated into components (e.g., in an acrylamide gel) but not obtained either as pure (e.g. lacking contaminating proteins, or chromatography reagents such as denaturing agents and polymers, e.g. acrylamide or agarose) substance, or solutions.
  • purified LTM preparations will lack any contaminating proteins from the same animal from which LTM is normally produced, as can be accomplished by recombinant expression of, for example, a human LTM protein in a non-human cell.
  • Full length proteins or fragments corresponding to one or more particular motifs and/or domains or to arbitrary sizes, for example, at least 5, 10, 25, 50, 75, 100, 125, 150 amino acids in length are within the scope of the present invention.
  • isolated LTM polypeptides can be encoded by all or a portion of a nucleic acid sequence shown in any of SEQ ID NOs: 1-X.
  • Isolated peptidyl portions of LTM proteins can be obtained by screening peptides recombinantly produced from the corresponding fragment of the nucleic acid encoding such peptides.
  • fragments can be chemically synthesized using techniques lcnown in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry.
  • an LTM polypeptide of the present invention may be arbitrarily divided into fragments of desired length with no overlap of the fragments, or preferably divided into overlapping fragments of a desired length. The fragments can be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments which can function as either agonists or antagonists of a wild-type (e.g., "authentic") LTM protein.
  • Recombinant polypeptides preferred by the present invention are encoded by a nucleic acid, which is at least 85% homologous and more preferably 90% homologous and most preferably 95%) homologous with an amino acid sequence of an LTM protein.
  • an LTM protein of the present invention is a mammalian LTM protein. It will be understood that certain post-translational modifications, e.g., phosphorylation and the like, can increase the apparent molecular weight of the LTM protein relative to the unmodified polypeptide chain.
  • an LTM agonist mimetic
  • an LTM antagonist an LTM antagonist
  • Homologs of each of the subject LTM proteins can be generated by mutagenesis, such as by discrete point mutation(s), or by truncation. For instance, mutation can give rise to homologs which retain substantially the same, or merely a subset, of the biological activity of the LTM polypeptide from which it was derived.
  • antagonistic forms of the protein can be generated which are able to inhibit the function of the naturally occurring form of the protein, such as by competitively binding to a downstream or upstream member of the LTM cascade which includes the LTM protein.
  • agonistic forms of the protein may be generated which are constitutively active.
  • the LTM protein and homologs thereof provided by the subject invention may be either positive or negative regulators of memory consolidation.
  • the assays of the invention may include cells transfected to express a recombinant form of the subject LTM polypeptides.
  • the host cell may be any prokaryotic or eukaryotic cell.
  • a nucleotide sequence derived from the cloning of mammalian LTM proteins, encoding all or a selected portion of the full-length proteir can be used to produce a recombinant form of an LTM polypeptide via microbial or eukaryotic cellular processes.
  • LTM genes and/or LTM gene products are used for carrying out assays designed to identify agents which, by modulating the function of one or more of the LTM genes, can be used to modify long term memory consolidation in animals.
  • test agents can be assessed in a cell-based or cell-free assay for ability to inhibit or potentiate the activity of an LTM protein.
  • the LTM genes can range from cell surface receptors and secreted proteins to transcription factors.
  • the invention contemplates such drug-screening formats which detect compounds that, e.g., modulate an enzymatic activity of the LTM protein, modulate the half-life of the LTM protein, modulate the interaction of the LTM protein with other proteins, nucleic acids, carbohydrates or other biological molecules, modulate the cellular localization of the LTM protein and the like.
  • assay formats will suffice and, in light of the present inventions, will be comprehended by a skilled artisan.
  • agents which can be tested in the subject drug screening assays include small organic molecules, e.g., having a molecular weight less than 2500 amu, more preferably less than less than 1000, 750 or 500 amu. Such molecules can include peptide and non-peptide moieties, nucleic acids, carbohydrates and the like. In many embodiments, it will be desirable to repeat the assay for a plurality of different test agents. For example, the subject assays can be repeated for at least 10 different test agents, and in other embodiments, for at least 100, or even at least 1000 different test agents.
  • Soluble proteins can be they cytoplasmic or extracellular, can be recombinantly expressed and at least partially purified, or provided as lysates, for use in cell-free assays.
  • Membrane-associated proteins can, in certain instances, be purified in detergent or liposomes, or isolated as part of a cell membrane fraction or organelle preparation.
  • a reaction mixture is generated including the LTM protein and one or more proteins (or nucleic acids) which interact with the LTM protein, such molecules being referred to herein as "LTM-interacting partners" or "LTM-IP".
  • LTM-IP include proteins that function upstream (including both activators and repressors of LTM activity), and proteins or nucleic acids which function downstream of the LTM polypeptide, whether they are positively or negatively regulated by it.
  • the reaction mixture also includes one or more test compounds.
  • Detection and quantification of complexes of the LTM protein with upstream or downstream LTM-IP provide a means for determining a compound's efficacy at inhibiting or potentiating complex formation between LTM and the LTM- IPs.
  • the efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the test compound.
  • a control assay can also be performed to provide a baseline for comparison. In one control assay, isolated and purified LTM polypeptide is added to a composition containing the LTM-IP, and the formation of a complex is quantitated in the absence of the test compound.
  • LTM polypeptide and a binding partner may be detected by a variety of techniques. Modulation of the formation of complexes can be quantitated using, for example: detectably labeled proteins such as radiolabeled, fluorescently labeled, or enzymatically labeled proteins; by immunoassay; or by chromatographic detection. Typically, it will be desirable to immobilize either LTM or its interacting partner to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of the LTM protein to an upstream or downstream element, in the presence and absence of a candidate agent, can be accomplished in any vessel suitable for containing the reactants.
  • a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix.
  • glutathione-S-transferase/LTM (GST/LTM) fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with a cell lysate or other preparation including the LTM-IP and the test compound, and the mixture incubated under conditions conducive to complex formation (in the absence of the test compound), e.g. at physiological conditions for salt and pH, though slightly more stringent conditions may be desired.
  • the beads are washed to remove any unbound LTM-IP, and the matrix immobilized and the amount of LTM-IP in the matrix determined, or in the supernatant after the complexes are subsequently dissociated.
  • the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of LTM-IP found in the bead fraction quantitated from the gel using standard electrophoretic techniques.
  • LTM or its cognate binding partner can be immobilized utilizing conjugation of biotin and streptavidin.
  • biotinylated LTM proteins can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • antibodies reactive with the LTM protein can be derivatized to the wells of the plate, and the LTM protein trapped in the wells by antibody conjugation.
  • preparations of an LTM-IP and a test compound are incubated in the LTM-presenting wells of the plate, and the amount of complex trapped in the well can be quantitated.
  • Exemplary methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the LTM binding partner, or which are reactive with the LTM protein and compete with the binding partner; as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the binding partner, either intrinsic or extrinsic activity.
  • the enzyme can be chemically conjugated or provided as a fusion protein with an LTM- IP.
  • the LTM-IP can be chemically cross-linked or genetically fused with horseradish peroxidase, and the amount of polypeptide trapped in the complex can be assessed with a chromogenic substrate of the enzyme, e.g.
  • a fusion protein comprising the polypeptide and glutathione-S-transferase can be provided, and complex formation quantitated by detecting the GST activity using l-chloro-2,4-dinitrobenzene (Habig et al (1974) J. Biol. Chem. 249:7130).
  • the protein to be detected in the complex can be "epitope tagged" in the form of a fusion protein which includes a second polypeptide sequence for which antibodies are readily available (e.g. from commercial sources).
  • the GST fusion proteins described above can also be used for quantification of binding using antibodies against the GST moiety.
  • Other useful epitope tags include mycepitopes (e.g., see Ellison et al. (1991) J. Biol. Chem.
  • cell-free ebodiments include assays which detect an intrinsic activity of an LTM protein or a complex including an LTM protein, and identify compounds that increase or inhibit that activity.
  • the reaction micture can be generated to include the LTP protein, a substrate for an enzymatic activity of the LTM protein, and the test agent.
  • the rate of conversion of the substrate to product is determined, and can be compared to such control samples as the LTM proteins and substrate admixed alone.
  • Test agents which are inhibitors of the LTM activity will decrease the rate of conversion of the substrate to product, whereas test agents that increase that rate are likely to be agonists of the LTM activity.
  • the substrate is readily detectable, e.g., the conversion of substrate to product a colorimetric or fluorometric change in the reaction mixture which is detectable by spectroscopic means, or creates or destroys an epitope which is detectable by immunoassay.
  • the readily available LTM proteins provided by the present invention also facilitates the generation of cell- based assays for identifying small molecule agonists/antagonists and the like.
  • the ability of a test agent to alter the activitiy of an LTM protein in the cell may include directly detecting the formation of complexes including the LTM protein, detecting an intrinic enzymatic activity of the LTM protein, directly detecting a change in cellular localization of the LTM protein, detecting a post-translational modification to the LTM protein or a change in the stability of the LTM protein, or detecting the downstream consequence of any one of such events.
  • Such assays can be simple binding assays.
  • the assay can be used to identify compounds which bind to the receptor or effect the ability of the receptor to bind its ligand.
  • cells which are phenotypically sensitive to the presence or activity of the LTM protein e.g., if it produces a morphological change in the cell, can be caused to over- or under-express a recombinant LTM protein in the presence and absence of a test agent of interest, with the assay scoring for modulation in LTM responses by the target cell which mediated by the test agent.
  • agents which produce a statistically significant change in LTM-dependent responses can be identified.
  • the level of expression of genes or gene products which are up- or down-regulated in response to the presence or activity of an LTM protein can be detected.
  • the regulatory regions of such genes e.g., the 5' flanking promoter and enhancer regions, are operably linked to a detectable marker (such as luciferase) which encodes a gene product that can be readily detected.
  • a transcriptional based assay could be used, for example, in which an LTM-responsive regulatory sequence is operably linked to a detectable marker gene.
  • the subject drug screening assays can utilized the LTM proteins to generate a "two hybrid" assay (see, for example, U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al.
  • the two hybrid assay relies on reconstituting in vivo a functional transcriptional activator protein from two separate fusion proteins.
  • the method makes use of chimeric genes which express hybrid proteins.
  • a first cliimeric gene can be generated with the coding sequence for a DNA-binding domain of a transcriptional activator fused in frame to the coding sequence for an LTM protein.
  • the second hybrid protein encodes a transcriptional activation domain fused in frame to another polypeptide, e.g., and LTM-IP, which binds to the LTM protein. If the two fusion proteins are able to interact, e.g., form an LTM-dependent complex, they bring into close proximity the two domains of the transcriptional activator. This proximity is sufficient to cause transcription of a reporter gene which is operably linked to a transcriptional regulatory site which is bound by the DNA-binding domain of the first fusion proteins, and expression of the reporter gene can be detected and used to score for the interaction of the LTM and sample proteins.
  • LTM proteins GluRl and GluR2
  • the subject assays are used to identify compouds which modulate the activity of a glutamate receptor, such as GluRl (SEQ ID Nos. 1 and 2) or GluR2 (SEQ ID Nos. 3 and 4).
  • a glutamate receptor such as GluRl (SEQ ID Nos. 1 and 2) or GluR2 (SEQ ID Nos. 3 and 4).
  • the subject assays can be used to identifying agents which bind to the receptors and, e.g., mimic or potentiate the activity of the natural ligand, or which inhibit binding or signal transduction by the receptor.
  • test agents can be tested for competition with binding.
  • [ 3 H] AMP A binding can be assessed as follows: cells expressing GluRl or GluR2 are preincubated in 50 mM Tris-HCl buffer, pH 7.4, for 30 min, and then incubated at 4°C for 4h in 100 mM buffer plus 100 mM KCSN, 70 nM [ ⁇ ]AMPA (53 Ci/mmol, NEN, Boston, MA) and the test agent.
  • Binding of the radiolabeled AMP A is assessed and compared to the level of binding in the absence of the test compound. Nonspecific binding is measured in the presence of 1 mM L-glutamate.
  • the GluRl and GluR2 receptors appears to be coupled to postsynaptic inositol phosphate metabolism, e.g., activating the PLC/PKC/CREB/MapK pathway, as well as cAMP-mediated pathways. Accordingly, generation of second messengers, and regulation of gene expression can be used to asses the ability of a test agent to inhibit or potentiate the activity of GluRl or GluR2.
  • Exemplary LTM proteins Insulin-like Growth Factor 1
  • IGF1 (SEQ ID No.s 7 and 8) is an extracellular protein which mediates its inductive effect on cells at least in part through binding to the IGF1 receptor (IGF1-R).
  • the receptor has an intrinsic tyrosine kinase activity, which can be targeted in the subject assay for identifying inhibitors of the kinase activity.
  • Signal transduction from the receptor includes interaction with a series of proteins containing Src homology-2 (SH2) domains, including SH2-containing protein tyrosine phosphatase 2 (SHP-2) and SHC.
  • SH2 SH2-containing protein tyrosine phosphatase 2
  • SHP-2 SH2-containing protein tyrosine phosphatase 2
  • the subject assay can be used to identify agents which inhibit the interaction of IGF1-R with SH2 domains, or which inhibit the enzymatic activity of an SH2 protein which interacts with IGF1-R.
  • the assay can combine the tyrosine-phosphorylated IGF1-R ⁇ -subunit, at least the SH2 domain of SHP-2, and a test agent, and detect the ability of the test agent to alter the formation of complexes containing IGF1-R and SHP-2.
  • IGF1-R also signals through acitvation of the Jak protein tyrosine kinase family, with resultant phosphorylation of the STAT transcriptional activator factors. See, for example, Frank et al (1995) PNAS 92:7779-7783; Scharfe et al. (1995) Blood 86:2077- 2085; Bacon et al. (1995) PNAS 92:7307-7311); and Sakatsume et al (1995) J Biol Chem 270:17528-17534. Events downstream of Jak phosphorylation have also been elucidated.
  • IGF1-R can cause the phosphorylation of signal transducers and activators of transcription (STAT) proteins STATl D, STAT2 ⁇ , and STAT3, as well as of two STAT-related proteins, p94 and p95.
  • STAT proteins translocate to the nucleus and bind to specific DNA sequences, thus suggesting a mechanism by which IGF1-R may activate specific genes
  • Detection means which may be scored for in the present assay, in addition to direct detection of second messangers, such as by changes in phosphorylation, includes reporter constructs or indicator genes which include transcriptional regulatory elements responsive to the STAT proteins.
  • reporter constructs or indicator genes which include transcriptional regulatory elements responsive to the STAT proteins.
  • Exemplary LTM proteins neuroendocrine VGF
  • VGF neurotropin-inducible gene
  • SEQ ID Nos. 11 and 12 The neurotropin-inducible gene VGF (SEQ ID Nos. 11 and 12) is expressed in neuronal and endocrine tissues. It encodes a secretory protein that is proteolytically processed in neuronal cells to low molecular mass polypeptides.
  • the subject assays can also be used to identify agents which inhibit the proteolytic processing of VGF.
  • Zif268 (SEQ ID Nos. 5 and 6) is also known as lcrox-24, egr-1, TIS 8, NGFI-A or zenk. It is a zinc-finger transcription factor which binds to a so-called "EGRl motif, e.g., atrascriptional regulatory sequence of 5'-CGCCCCCGC or 5'-GCGTGGGCG. See, for example, Rauscher et al. (1990) Science 250:1259; and Pavletich et al. (1991) Science 252:809.
  • the subject assay can be a transcription based assay including a reporter gene having an EGRl motif as part of its transcriptional regulatory sequences.
  • Test agents can be assessed for their ability to enhance or inhibit Zif268- dependent transcription.
  • test agents can be tested for their ability to ehance or inhibit binding of Zif268 with its EGRl motif in a competitive binding assay, e.g, a nucleic acid including the motif.
  • a competitive binding assay e.g, a nucleic acid including the motif.
  • the target for the subject drug screening assay is the transcription factor C/EBP ⁇ (SEQ ID Nos. 9 and 10), also referred to as the CCAAT/enhancer protein.
  • the CCAAT/Enhancer Binding Protein (C/EBP) family belongs to the basic leucine zipper class of transcription factors.
  • the C/EBP protein binds to the CCAAT-box (consensus GG T / C CAATCT).
  • C/EBP-dependent transcriptional activity or competition binding assays can be used to assessed the ability of test compounds to enhance or inhibit C/EBP ⁇ activity.
  • Second Messenger Generation When screening for bioactivity of test compounds, intracellular second messenger generation can be measured directly.
  • a variety of intracellular effectors have been identified as being regulated by certain of the LTM proteins described above, including adenylyl cyclase, cyclic GMP, phosphodiesterases, phosphoinositidase C, and phospholipase A2, as well as a variety of ions.
  • the detection signal is provided by detecting phosphorylation of intracellular proteins, e.g., MEKKs, MEKs, or Map kinases, or by the use of reporter constructs or indicator genes which include transcriptional regulatory elements responsive to c-fos and/or c-jun.
  • the GTPase enzymatic activity by G proteins can be measured in plasma membrane preparations by determining the breakdown of D ⁇ 2p GTP using techniques that are lcnown in the art (For example, see Signal Transduction: A Practical Approach. G. Milligan, Ed. Oxford University Press, Oxford England).
  • receptors that modulate cAMP are tested, it will be possible to use standard techniques for cAMP detection, such as competitive assays which quantitate [ ⁇ HJcAMP in the presence of unlabelled cAMP.
  • LTM proteins stimulate the activity of phospholipase C which stimulates the breakdown of phosphatidylinositol 4,5, bisphosphate to 1,4,5-IP3 (which mobilizes intracellular Ca++) and diacylglycerol (DAG) (which activates protein kinase C).
  • Inositol lipids can be extracted and analyzed using standard lipid extraction techniques. DAG can also be measured using thin-layer chromatography. Water soluble derivatives of all three inositol lipids (IP1, IP2, IP3) can also be quantitated using radiolabelling techniques or HPLC.
  • DAG can also be produced from phosphatidyl choline.
  • the breakdown of this phospholipid in response to receptor- mediated signaling can also be measured using a variety of radiolabelling techniques.
  • the activation of phospholipase A2 can easily be quantitated using lcnown techniques, including, for example, the generation of arachadonate in the cell.
  • receptors such as IGF1-R
  • assay formats may be useful when the receptor of interest is a receptor kinase or phosphatase.
  • immunoblotting (Lyons and Nelson (1984) Proc. Natl. Acad. Sci. USA 81:7426-7430) using anti- phosphotyrosine, anti-phosphoserine or abti-phosphothreonine antibodies.
  • tests for phosphorylation could be also useful when the receptor itself may not be a lcinase, but activates protein kinases or phosphatase that function downstream in the signal transduction pathway.
  • MAP kinase pathway that appears to mediate both mitogenic, differentiation and stress responses in different cell types. Stimulation of growth factor receptors results in Ras activation followed by the sequential activation of c-Raf, MEK, and p44 and p42 MAP kinases (ERKl and ERK2). Activated MAP Icinase then phosphorylates many key regulatory proteins, including p90RSK and Elk-1 that are phosphorylated when MAP kinase translocates to the nucleus. Homologous pathways exist in mammalian and yeast cells. For instance, an essential part of the S.
  • cerevisiae pheromone signaling pathway is comprised of a protein kinase cascade composed of the products of the STE11, STE7, and FUS3/KSS1 senes (the latter pair are distinct and functionally redundant). Accordingly, phosphorylation and/or activation of members of this kinase cascade can be detected and used to quantitate receptor engagement.
  • Phosphotyrosine specific antibodies are available to measure increases in tyrosine phosphorylation and phospho-specific antibodies are commercially available (New England Biolabs, Beverly, MA).
  • the signal transduction pathway of the LTM protein upregulates expression or otherwise activates an enzyme which is capable of cleaving a substrate which can be added to the cell.
  • the signal can be detected by using a detectable substrate, in which case lose of the substrate signal is monitored, or alternatively, by using a substrate which produces a detectable product.
  • the conversion of the substrate to product by the activated enzyme produces a detectable change in optical characteristics of the test cell, e.g., the substrate and/or product is chromogenically or fluorogenically active.
  • the signal transduction pathway causes a change in the activity of a proteolytic enzyme, altering the rate at which it cleaves a substrate peptide (or simply activates the enzyme towards the substrate).
  • the peptide includes a fluorogenic donor radical, e.g., a fluorescence emitting radical, and an acceptor radical, e.g., an aromatic radical which absorbs the fluorescence energy of the fluorogenic donor radical when the acceptor radical and the fluorogenic donor radical are covalently held in close proximity. See, for example, USSN 5,527,681, 5,506,115, 5,429,766, 5,424,186, and 5,316,691; and Capobianco et al.
  • the substrate peptide has a fluorescence donor group such as 1-aminobenzoic acid (anthranilic acid or ABZ) or aminomethylcoumarin (AMC) located at one position on the peptide and a fluorescence quencher group, such as lucifer yellow, methyl red or nitrobenzo-2-oxo- 1,3-diazole (NBD), at a different position near the distal end of the peptide.
  • a cleavage site for the activated enzyme will be diposed between each of the sites for the donor and acceptor groups.
  • the intramolecular resonance energy transfer from the fluorescence donor molecule to the quencher will quench the fluorescence of the donor molecule when the two are sufficiently proximate in space, e.g., when the peptide is intact.
  • the quencher is separated from the donor group, leaving behind a fluorescent fragment.
  • activation of the enzyme results in cleavage of the detection peptide, and dequenching of the fluorescent group.
  • the detectable signal can be produced by use of enzymes or chromogenic/fluorscent probes whose activities are dependent on the concentration of a second messanger, e.g., such as calcium, hydrolysis products of inositol phosphate, cAMP, etc.
  • a second messanger e.g., such as calcium, hydrolysis products of inositol phosphate, cAMP, etc.
  • the mobilization of intracellular calcium or the influx of calcium from outside the cell can be measured using standard techniques.
  • the choice of the appropriate calcium indicator, fluorescent, bioluminescent, metallochromic, or Ca++-sensitive microelectrodes depends on the cell type and the magnitude and time constant of the event under study (Borle (1990) Environ Health Perspect 84:45-56).
  • Ca++ detection cells could be loaded with the Ca++sensitive fluorescent dye fura-2 or indo-1, using standard methods, and any change in Ca++ measured using a fluorometer.
  • the signal transduction activity of a receptor or ion channel pathway can be measured by detection of a transcription product, e.g., by detecting receptor/channel-mediated transcriptional activation (or repression) of a gene(s).
  • Detection of the transcription product includes detecting the gene transcript, detecting the product directly (e.g., by immunoassay) or detecting an activity of the protein (e.g., such as an enzymatic activity or chromogenic/fluorogenic activity); each of ' which is generally referred to herein as a means for detecting expression of the indicator gene.
  • the indicator gene may be an unmodified endogenous gene of the host cell, a modified endogenous gene, or apart of a completely heterologous construct, e.g., as part of a reporter gene construct.
  • the indicator gene is an unmodified endogenous gene.
  • the transcriptional activation ability of the signal pathway can be amplified by the overexpression of one or more of the proteins involved in the intracellular signal cascade, particularly enzymes involved in the pathway. For example, increased expression of Jun kinases (JNKs) can potentiate the level of transcriptional activation by a signal in an MEK/MEKK pathway. This approach can, of course, also be used to potentiate the level of transcription of a heterologous reporter gene as well.
  • JNKs Jun kinases
  • the sensitivity of an endogenous indicator gene can be enhanced by manipulating the promoter sequence at the natural locus for the indicator gene. Such manipulation may range from point mutations to the endogenous regulatory elements to gross replacement of all or substantial portions of the regulatory elements.
  • manipulation of the genomic sequence for the indicator gene can be carried out using techniques known in the art, including homologous recombination.
  • the promoter (or other transcriptional regulatory sequences) of the endogenous gene can be "switched out" with a heterologous promoter sequence, e.g., to form a chimeric gene at the indicator gene locus.
  • a heterologous promoter sequence e.g., to form a chimeric gene at the indicator gene locus.
  • the regulatory sequence can be so altered at the genomic locus of the indicator gene.
  • a heterologous reporter gene construct can be used to provide the function of an indicator gene.
  • Reporter gene constructs are prepared by operatively linking a reporter gene with at least one transcriptional regulatory element. If only one transcriptional regulatory element is included it must be a regulatable promoter, At least one the selected transcriptional regulatory elements must be indirectly or directly regulated by the activity of the selected cell-surface receptor whereby activity of the receptor can be monitored via transcription of the reporter genes.
  • Many reporter genes and transcriptional regulatory elements are known to those of skill in the art and others may be identified or synthesized by methods known to those of skill in the art. g. Exemplary Screening and Selection Assays: Reporter Genes
  • reporter genes include, but are not limited to CAT (chloramphenicol acetyl transferase) (Alton and Vapnelc (1979), Nature 282: 864-869) luciferase, and other enzyme detection systems, such as beta-galactosidase; firefly luciferase (deWet et al. (1987), Mol. Cell. Biol. 7:725-737); bacterial luciferase (Engebrecht and Silverman (1984), PNAS 1 : 4154-4158; Baldwin et al. (1984), Biochemistry 23: 3663-3667); alkaline phosphatase (Toh et al. (1989) Eur. J. Biochem.
  • CAT chloramphenicol acetyl transferase
  • Transcriptional control elements for use in the reporter gene constructs, or for modifying the genomic locus of an indicator gene include, but are not limited to, promoters, enhancers, and repressor and activator binding sites.
  • Suitable transcriptional regulatory elements may be derived from the transcriptional regulatory regions of genes whose expression is rapidly induced, generally within minutes, of contact between the cell surface protein and the effector protein that modulates the activity of the cell surface protein. Examples of such genes include, but are not limited to, the immediate early genes (see, Sheng et al. (1990) Neuron 4: 477-485), such as c-fos.
  • Immediate early genes are genes that are rapidly induced upon binding of a ligand to a cell surface protein.
  • the transcriptional control elements that are preferred for use in the gene constructs include transcriptional control elements from immediate early genes, elements derived from other genes that exhibit some or all of the characteristics of the immediate early genes, or synthetic elements that are constructed such that genes in operative linkage therewith exhibit such characteristics.
  • the characteristics of preferred genes from which the transcriptional control elements are derived include, but are not limited to, low or undetectable expression in quiescent cells, rapid induction at the transcriptional level within minutes of extracellular simulation, induction that is transient and independent of new protein synthesis, subsequent shut-off of transcription requires new protein synthesis, and mRNAs transcribed from these genes have a short half-life. It is not necessary for all of these properties to be present.
  • VIP vasoactive intestinal peptide
  • somatostatin cAMP responsive; Montminy et al. (1986), Proc. Natl. Acad. Sci. 8.3:6682-6686
  • proenkephalin promoter responsive to cAMP, nicotinic agonists, and phorbol esters; Comb et al.
  • a transcriptional based readout can be constructed using the cyclic AMP response element binding protein, CREB, which is a transcription factor whose activity is regulated by phosphorylation at a particular serine (SI 33).
  • CREB cyclic AMP response element binding protein
  • SI 33 serine
  • CREB binds to a recognition sequence lcnown as a CRE (cAMP Responsive Element) found to the 5' of promotors known to be responsive to elevated cAMP levels.
  • CRE cAMP Responsive Element
  • Phosphorylation of CREB is seen in response to both increased cAMP levels and increased intracellular Ca levels. Increased cAMP levels result in activation of PKA, which in turn phosphorylates CREB and leads to binding to CRE and transcriptional activation. Increased intracellular calcium levels results in activation of calcium/calmodulin responsive kinase IV (CaM kinase IV). Phosphorylation of CREB by CaM kinase IV is effectively the same as phosphorylation of CREB by PKA, and results in transcriptional activation of CRE containing promotors. Activation of extracellular signal-related protein kinase (ERK) and Rsk2 by also leads to the phosphorylation and transactivation of CREB. Impey et al. (1998) Neuron 21:869-883.
  • a transcriptional-based readout can be constructed in cells containing a reporter gene whose expression is driven by a basal promoter containing one or more CRE. Changes in the intracellular concentration of Ca ++ (a result of alterations in the activity of the receptor upon engagement with a ligand) will result in changes in the level of expression of the reporter gene if: a) CREB is also co-expressed in the cell, and b) either the endogenous yeast CaM kinase will phosphorylate CREB in response to increases in calcium or if an exogenously expressed CaM kinase IV is present in the same cell. In other words, stimulation of PLC activity will result in phosphorylation of CREB and increased transcription from the CRE-construct, while inhibition of PLC activity will result in decreased transcription from the CRE-responsive construct.
  • a reporter construct for use in the present invention for detecting signal transduction through STAT proteins can be generated by using -71 to +109 of the mouse c-fos gene fused to the bacterial • chloramphenicol acetyltransferase gene (-71fosCAT) or other detectable marker gene.
  • Induction by a cytokine receptor induces the tyrosine phosphorylation of STAT and STAT-related proteins, with subsequent translocation and binding of these proteins to the STAT-RE. This then leads to activation of transcription of genes containing this DNA element within their promoters.
  • the reporter gene is a gene whose expression causes a phenotypic change which is screenable or selectable. If the change is selectable, the phenotypic change creates a difference in the growth or survival rate between cells which express the reporter gene and those which do not. If the change is screenable, the phenotype change creates a difference in some detectable characteristic of the cells, by which the cells which express the marker may be distinguished from those which do not. Selection is preferable to screening in that it can provide a means for amplifying from the cell culture those cells which express a test polypeptide which is a receptor effector.
  • the marker gene is coupled to the receptor signaling pathway so that expression of the marker gene is dependent on activation of the receptor. This coupling may be achieved by operably linking the marker gene to a receptor-responsive promoter.
  • receptor-responsive promoter indicates a promoter which is regulated by some product of the target receptor's signal transduction pathway.
  • the promoter may be one which is repressed by the receptor pathway, thereby preventing expression of a product which is deleterious to the cell.
  • a receptor repressed promoter one screens for agonists by linking the promoter to a deleterious gene, and for antagonists, by linking it to a beneficial gene.
  • Repression may be achieved by operably linking a receptor- induced promoter to a gene encoding mRNA which is antisense to at least a portion of the mRNA encoded by the marker gene (whether in the coding or flanking regions), so as to inhibit translation of that mRNA.
  • Repression may also be obtained by linking a receptor-induced promoter to a gene encoding a DNA binding repressor protein, and incorporating a suitable operator site into the promoter or other suitable region of the marker gene.
  • a receptor-induced promoter to a gene encoding a DNA binding repressor protein
  • incorporating a suitable operator site into the promoter or other suitable region of the marker gene.
  • Suitable host cells for generating the subject assay include prokaryotes, yeast, or higher eukaryotic cells, especially mammalian cells.
  • Prokaryotes include gram negative or gram positive organisms.
  • suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman (1981) Cell 23:175) CV-1 cells (ATCC CCL 70), L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa and BHK cell lines. It will be understood that to achieve selection or screening, the host cell must have an appropriate phenotype.
  • yeast cells may be of any species which are cultivable and in which an exogenous receptor can be made to engage the appropriate signal transduction machinery of the host cell. Suitable species include Kluyverei lactis, Schizosaccharomyces pombe, and Ustilaqo maydis; Saccharomyces cerevisiae is preferred. Other yeast which can be used in practicing the present invention are Neurospora crassa, Aspergillus niger, Aspergillus nidulans, Pichia pastoris, Candida tropicalis, and Hansenula polymorpha.
  • yeast includes not only yeast in a strictly taxonomic sense, i.e., unicellular organisms, but also yeast-like multicellular fungi or filamentous fungi.
  • reporter constructs can provide a selectable or screenable trait upon transcriptional activation (or inactivation) in response to a signal transduction pathway coupled to LTM protein of interest.
  • the reporter gene may be an unmodified gene already in the host cell pathway, such as the genes responsible for growth arrest in yeast. It may be a host cell gene that has been operably linked to a "receptor-responsive" promoter. Alternatively, it may be a heterologous gene (e.g., a "reporter gene construct" that has been so linked. Suitable genes and promoters are discussed below.
  • second messenger generation can be measured directly in the detection step, such as mobilization of intracellular calcium or phospholipid metabolism are quantitated.
  • indicator genes can be used to detect receptor-mediated signaling.
  • such compounds can be tested in an animal model for studying fornix-mediated memory consolidation, e.g., an animal which as been prepared by introducing lesions to the formix structure.
  • an animal model for studying fornix-mediated memory consolidation e.g., an animal which as been prepared by introducing lesions to the formix structure.
  • animals with fornix lesions learn the inhibitory avoidance and display memory at control levels for up to 6 hours, however, by 24 hours they exhibit amnesia.
  • the subject method utilizes an animal which has been manipulated to create at least partial disruption of fornix-mediated signalling to the hippocampus, the disruption affecting memory consolidation and learned behavior in the animal.
  • the animal is conditioned with a learning or memory regimen which results in learned behavior in the mammal in the absence of the fornix lesion.
  • Test agents are administered to the animal in order assess their effects on memory consolidation. An increase in learned behavior, relative to the absence of the test agent, indicates that the test agent enhances memory consolidation.
  • retention of the learned behavior can be determined, for example, after at least about 12- 24 hours, 14-22 hours, 16-20 hours and or 18-19 hours after completion of the learning phase to determine whether the agent promotes memory consolidation. In a particular embodiment, retention of the learned behavior can be determined 24 hours after completion of the learning phase.
  • the lesion mammal can have a lesion of the fornix or a related brain structure that disrupts memory consolidation (e.g., perirhinal cortex, amygdala, medial septal nucleus, locus coeruleus, hippocampus, mammallary bodies). Lesions in the mammal can be produced by mechanical or chemical disruption.
  • the fornix lesion can be caused by surgical ablation, electrolytic, neurotoxic and other chemical ablation techniques, or reversible inactivation such as by injection of an anesthetic, e.g., tetrodooxin or lidocaine, to temporarily arrest activity in the fornix.
  • anesthetic e.g., tetrodooxin or lidocaine
  • fimbrio-fomix (rodents) and fornix (primates) lesions can be created by stereotatic ablation.
  • neurons of the fornix structure are axotomized, e.g., by transection or aspiration (suction) ablation.
  • a complete transection of the fornix disrupts cholinergic and GABAergic function and electrical activity, and induces morphological reorganization in the hippocampal formation.
  • the fornix transection utilized in the subject method will not disconnect the parahippocampal region from the neocortex.
  • the fornix transection will not disrupt functions that can be carried out by the parahippocampal region independent of processing by the hippocampal formation, and hence would not be expected to produce the full-blown amnesia seen following more complete hippocampal system damage.
  • the animal can be a rat.
  • the animals are anesthetized, e.g., with intraperitoneal injections of a lcetamine-xylazine mixture and positioned in a Kopf stereotoxic instrument.
  • a sagittal incision is made in the scalp and a craniotomy is performed extending 2.0 mm posterior and 3.0 mm lateral from Bregma.
  • An aspirative device e.g., with a 20 gauge tip, is mounted to a stereotaxic frame (Kopf Instruments) and fimbria-fornix is aspirated by placing the suction tip at the correct sterotaxic location in the animals brain.
  • Unilateral aspirative lesions are made by suction through the cingulate cortex, completely transecting the fimbria fornix unilaterally, and (optionally) removing the dorsal tip of the hippocampus as well as the overlying cingulate cortex to inflict a partial denervation on the hippocampus target. See also, Gage et al., (1983) Brain Res. 268:27 and Gage et al. (1986) Neuroscience 19:241.
  • the animal can be a monkey.
  • the animals can be anesthetized, e.g., with isoflurane (1.5-2.0%).
  • isoflurane (1.5-2.0%).
  • mannitol (0.25 g/kg, iv)
  • unilateral transections of the left fornix an be performed, such as described by Kordower et al. (1990) J. Comp. Neurol.. 298:443.
  • a surgical drill is used to create a parasagittal bone flap which exposes the frontal superior sagittal sinus.
  • the dura is retracted and a self-retaining retractor is used to expose the interhemispheric fissure.
  • the corpus callosum is longitudinally incised.
  • the fornix is easily visualized as a discrete 2-3 mm wide white fiber bundle.
  • the fornix can be initially transected using a ball dissector. The cut ends of the fornix can then be suctioned to ensure completeness of the lesion.
  • the fornix lesion can be created by excitotoxically, or by other chemical means, inhibiting or ablating fornix neurons, or the cells of the hippocampus which are innervated by fornix neurons.
  • the fornix lesion is generated by selective disruption of particular neuronal types, such as fornix cholinergic, GABAergic and/or serotonergic neurons, and in certain embodiments, particular morphological subtypes within such neuron types.
  • selective ablation of serotonergic neurons can be accomplished by treatment of the fornix structure with methamphetamines, such as d-methamphetamine (d-MA), methylenedioxyamphetamine (MDA) and methylenedioxymethamphetamine (MDMA), and 5,7-dihydroxytryptamine (5,7-DHT).
  • methamphetamines such as d-methamphetamine (d-MA), methylenedioxyamphetamine (MDA) and methylenedioxymethamphetamine (MDMA), and 5,7-dihydroxytryptamine (5,7-DHT).
  • d-MA d-methamphetamine
  • MDA methylenedioxyamphetamine
  • MDMA methylenedioxymethamphetamine
  • 5,7-DHT 5,7-dihydroxytryptamine
  • the afferant fornix signals to the hippocampus due to cholmergic neurons can be ablated
  • the agents such as 6-OHDA and ibotenic acid can be used to selectively destroy fornix dopamine neurons as part of the ablative regimen.
  • Other exemplary agents which may used to create fornix lesions include N-rnethyl-D-aspartate (NMD A), quinolinic acid, and methylazoxymethanol.
  • Learning and/or memory tests include, for example, inhibitory avoidance, contextual fear conditioning, visual delay non-match to sample, spatial delay non-match to sample, visual discrimination, Barnes circular maze, Morris water maze and Radial arm maze tests.
  • An exemplary inhibitory avoidance test utilizes an apparatus that consists of a lit chamber that can be separated from a dark chamber by a sliding door.
  • the animal is placed in the lit chamber for some period of time, then the door is opened, the animal moves to the dark chamber after a short delay-the latency, that is recorded.
  • the door is shut closed and a footshock is delivered. Retention of the experience is determined after various time intervals, e.g., 24 or 48 hours, by repeating the test and recording the latency.
  • the protocol is one of many variants of the inhibitory avoidance procedures (for review, see Rush (1988) Behav Neural Biol 50:255).
  • An exemplary maze testing embodiment is the water maze working memory test.
  • the method utilizes an apparatus which consists of a circular water tank.
  • the water in the tank is made cloudy by the addition of milk powder.
  • a clear plexiglass platform, supported by a movable stand rest on the bottom of the tank, is submerged just below the water surface.
  • Normally a swimming rat cannot perceive the location of the platform but it may recall it from a previous experience and training, unless it suffers from some memory impairment.
  • the time taken to locate the platform is measured and referred to as the latency.
  • all orientational cues such as ceiling lights etc. remain unchanged. Longer latencies are generally observed with rats with some impairment to their memory.
  • Another memory test includes the eyeblink conditioning test, which involves the administration of white noise or steady tone that preceeds a mild air puff which stimulates the subject's eyeblink.
  • Still another memory test which can be used is fear conditioning, e.g., either "cued” and “contextual” fear conditioning.
  • a freeze monitor administers a sequence of stimuli (sounds, shock) and then records a series of latencies measuring the recovery from shock induced freezing of the animal.
  • Another memory test for the lesioned animals is a holeboard test, which utilizes a rotating holeboard apparatus containing (four) open holes arranged in a 4-corner configuration in the floor of the test enclosure.
  • a mouse is trained to poke its head into a hole and retrieve a food reward from a "baited" hole which contains a reward on every trial.
  • There is a food reward e.g. Froot Loop
  • every exposed hole which is made inaccessible by being placed under a screen.
  • the screen allows the odor of the reward to emanate from the hole, but does not allow access to the reinforcer.
  • a small piece of Froot Loop is placed on top of the screen, where it is accessible.
  • the entire apparatus rests on a turntable so that it may be rotated easily to eliminate reliance on proximal (e.g. olfactory) cues.
  • a start tube is placed in the center of the apparatus. The subject is released from the tube and allowed to explore for the baited ("correct") hole.
  • agents identified in the subject assay can be formulated in pharmaceutically acceptable excipients for in vivo administration to an animal, preferably a human.
  • the compounds selected in the subject assay, or a pharmaceutically acceptable salt thereof may accordingly be formulated for administration with a biologically acceptable medium, such as water, buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures thereof.
  • a biologically acceptable medium such as water, buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures thereof.
  • a biologically acceptable medium includes any and all solvents, dispersion media, and the like which may be appropriate for the desired route of administration of the pharmaceutical preparation. The use of such media for pharmaceutically active substances is known in the art.
  • Suitable vehicles and their formulation inclusive of other proteins are described, for example, in the book Remington 's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences. Mack Publishing Company, Easton, Pa., USA 1985). These vehicles include injectable "deposit formulations". Based on the above, such pharmaceutical formulations include, although not exclusively, solutions or freeze-dried powders of the compound in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered media at a suitable pH and isosmotic with physiological fluids. In preferred embodiment, the compound can be disposed in a sterile preparation for topical and/or systemic administration.
  • excipients such as, but not exclusively, mannitol or glycine may be used and appropriate buffered solutions of the desired volume will be provided so as to obtain adequate isotonic buffered solutions of the desired pH.
  • Similar solutions may also be used for the pharmaceutical compositions of compounds in isotonic solutions of the desired volume and include, but not exclusively, the use of buffered saline solutions with phosphate or citrate at suitable concentrations so as to obtain at all times isotonic pharmaceutical preparations of the desired pH, (for example, neutral pH).
  • the pharmaceutical of the present invention is a gene delivery system for gene therapy with a therapeutic LTM gene.
  • gene therapy systems can be introduced into a patient by any of a number of methods, each of which is familiar in the art.
  • a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g. by intravenous injection, and specific transduction of the protein in the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the receptor gene, or a combination thereof.
  • initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized.
  • the gene delivery vehicle can be introduced by catheter (see U.S. Pat. No. 5,328,470) or by stereotactic injection (e.g. Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91: 3054-3057).
  • the pharmaceutical preparation of the gene therapy construct can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can comprise one or more cells which produce the gene delivery system.
  • the present invention contemplates modes of treatment and prophylaxis which utilitize one or more of the subject LTM genes (e.g., by gene therapy) or antisense constructs thereto, the LTM proteins (e.g., for protein therapy) or peptidomimetics thereof, or compounds identified in the subject drug screening assays.
  • LTM genes e.g., by gene therapy
  • antisense constructs e.g., the LTM proteins
  • peptidomimetics thereof e.g., for protein therapy
  • compounds identified in the subject drug screening assays e.g., for protein therapy
  • these agents may be useful for altering (increasing or decreasing) the occurrence of learning and/or memory defects in an organism, and thus, altering the learning ability and/or memory capacity of the organism.
  • the preparations of the present invention can be used simply to enhance normal memory function.
  • Memory disorders which can be treated according to the present invention may have a number of origins: a functional mechanism (anxiety, depression), physiological aging (age-associated memory impairment), drugs, or anatomical lesions (dementia). Indications for which such preparations may be useful include learning disabilities, memory impairment, e.g., due to toxicant exposure, brain injury, age, schizophrenia, epilepsy, mental retardation in children and senile dementia, including Alzheimer's disease.
  • the invention contemplates the treatment of amnesia.
  • Amnesias are described as specific defects in declarative memory. Faithful encoding of memory requires a registration, rehearsal, and retention of information. The first two elements appear to involve the hippocampus and medial temporal lobe structures. The retention or storage appears to involve the heteromodal association areas. Amnesia can be experienced as a loss of stored memory or an inability to form new memories. The loss of stored memories is lcnown as retrograde amnesia. The inability to form new memories is known as anterograde amnesia.
  • amnesias which may be treated by the subject method include, amensias of short duration, alcoholic blackouts, Wernicke-Korsakoff s (early), partial complex seizures, transient global amnesia, those which are medication related, such as triazolam (Halcion), basilar artery migraines.
  • the subject method may also be used to treat amensias of longer duration, such as post concussive or as the result of Herpes simplex encephalitis .
  • Toxicity and therapeutic efficacy of compounds to be used in the treatment methods of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining The LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50%) of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • the present method also provides a method for determining if a subject is at risk for a disorder characterized deterioration of memory consolidation.
  • the methods can be characterized as comprising detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of (i) an alteration affecting the integrity of a gene encoding an LTM protein, or (ii) the mis-expression of the LTM gene.
  • such genetic lesions can be detected by ascertaining the existence of at least one of (i) a deletion of one or more nucleotides from an LTM gene, (ii) an addition of one or more nuclectides to an LTM gene, (iii) a substitution of one or more nucleotides of an LTM gene, (iv) a gross chromosomal rearrangement of an LTM gene, (v) a gross alteration in the level of a messenger RNA transcript of an LTM gene, (vii) aberrant modification of an LTM gene, such as of the methylation pattern of the genomic DNA, (vii) the presence of a non-wild type splicing pattern of a messenger RNA transcript of an LTM gene, (viii) a non-wild type level of an LTM-protein, (ix) allelic loss of an LTM gene, and (x) inappropriate post-translational modification of an LTM-protein.
  • the present invention provides a large number of assay techniques for detecting lesions
  • a nucleic acid composition comprising a (purified) oligonucleotide probe including a region of nucleotide sequence which is capable of hybridizing to a sense or antisense sequence of an LTM gene, such as represented by any of SEQ ID NOs: 1, 3, 5, 7, 9 or 11, or naturally occurring mutants thereof, or 5' or 3' flanking sequences or intronic sequences naturally associated with the subject LTM genes or naturally occurring mutants thereof.
  • the nucleic acid of a cell is rendered accessible for hybridization, the probe is exposed to nucleic acid of the sample, and the hybridization of the probe to the sample nucleic acid is detected.
  • Such techniques can be used to detect lesions at either the genomic or mRNA level, including deletions, substitutions, etc., as well as to determine mRNA transcript levels.
  • the method can be generally characterized as comprising detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by an alteration affecting the integrity of an LTM gene.
  • a genetic lesion characterized by an alteration affecting the integrity of an LTM gene.
  • such genetic lesions can be detected by ascertaining the existence of at least one of (i) a deletion of one or more nucleotides from an LTM gene, (ii) an addition of one or more nucleotides to an LTM gene, (iii) a substitution of one or more nucleotides of an LTM gene, and (iv) the presence of a non-wild type splicing pattern of a messenger RNA transcript of an LTM gene.
  • detection of the lesion comprises utilizing the probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al.
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • the method includes the steps of (i) collecting a sample of cells from a patient, (ii) isolating nucleic acid (c .g., genomic, mRNA or both) from the cells of the sample, (iii) contacting the nucleic acid sample with one or more primers which specifically hybridize to an LTM gene under conditions such that hybridization and amplification of the LTM gene (if present) occurs, and (iv) detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample.
  • nucleic acid c .g., genomic, mRNA or both
  • contacting the nucleic acid sample with one or more primers which specifically hybridize to an LTM gene under conditions such that hybridization and amplification of the LTM gene (if present) occurs
  • detecting the presence or absence of an amplification product or detecting the size of the amplification product and comparing the length to a control sample.
  • PCR and/or LCR may
  • nucleic acid composition comprising a (purified) oligonucleotide probe including a region of nucleotide sequence which is capable of hybridizing to a sense or antisense sequence of an LTM gene, or naturally occurring mutants thereof, or 5' or 3' flanking sequences or intronic sequences naturally associated with the subject LTM genes or naturally occurring mutants thereof.
  • the nucleic acid of a cell is rendered accessible for hybridization, the probe is exposed to nucleic acid of the sample, and the hybridization of the probe to the sample nucleic acid is detected.
  • Such techniques can be used to detect lesions at either the genomic or mRNA level, including deletions, substitutions, etc., as well as to determine mRNA transcript levels.
  • Such oligonucleotide probes can be used for both predictive and therapeutic evaluation of allelic mutations which might be manifest in, for example, deterioration in memeory consolidation.
  • the methods described herein may be performed, for example, by utilizing prepackaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving memory or an LTM gene.
  • Antibodies directed against wild type or mutant LTM proteins which are discussed, above, may also be used in disease diagnostics and prognostics.
  • Such diagnostic methods may be used to detect abnormalities in the level of LTM protein expression, or abnormalities in the structure and/or tissue, cellular, or subcellular location of LTM protein.
  • Structural differences may include, for example, differences in the size, electronegativity, or antigenicity of the mutant LTM protein relative to the normal LTM protein.
  • Protein from the tissue or cell type to be analyzed may easily be detected or isolated using techniques which are well lcnown to one of skill in the art, including but not limited to western blot analysis.
  • western blot analysis For a detailed explanation of methods for carrying out western blot analysis, see Sambrook et al, 1989, supra, at Chapter 18.
  • the protein detection and isolation methods employed herein may also be such as those described in Harlow and Lane, for example, (Harlow, E. and Lane, D., 1988, “Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.), which is incorporated herein by reference in its entirety.
  • the antibodies (or fragments thereof) useful in the present invention may, additionally, be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of LTM proteins.
  • In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody of the present invention.
  • the antibody (or fragment) is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample.
  • a solid phase support or carrier is used as a support capable of binding an antigen or an antibody.
  • supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
  • the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody.
  • the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
  • the surface may be flat such as a sheet, test strip, etc.
  • Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
  • EIA enzyme immunoassay
  • the enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means.
  • Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha- glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • the detection can be accomplished by colorimetric methods which employ a
  • Detection may also be accomplished using any of a variety of other immunoassays.
  • a radioimmunoassay RIA
  • the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.
  • fluorescent labeling compounds fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • the antibody can also be detectably labeled using fluorescence emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTP A) or ethylenediaminetetraacetic acid (EDTA).
  • DTP A diethylenetriaminepentacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • the antibody also can be detectably labeled by coupling it to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a bioluminescent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in, which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.
  • Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
  • the cell- and animal-based model systems may be used to further characterize the role of the subject LTM genes and proteins in memory.
  • assays may be utilized as part of screening strategies designed to identify compounds which are capable of ameliorating disease symptoms.
  • the animal- and cell-based models may be used to identify drugs, pharmaceuticals, therapies and interventions which may be effective in treating disease.
  • transgenic animal which is comprised of cells (of that animal) which contain a transgene of the present invention and which preferably (though optionally) express an exogenous LTM protein in one or more cells in the animal.
  • An LTM transgene can encode the wild-type form of the protein, or can encode homologs thereof including both agonists and antagonists, as well as antisense constructs.
  • the expression of the transgene is restricted to specific subsets of cells, tissues or developmental stages utilizing, for example, cis-acting sequences that control expression in the desired pattern.
  • such mosaic expression of an LTM protein can be essential for many forms of lineage analysis and can additionally provide a means to assess the effects of, for example, lack of LTM expression which might grossly alter development in small patches of tissue within an otherwise normal embryo.
  • tissue-specific regulatory sequences and conditional regulatory sequences can be used to control expression of the transgene in certain spatial patterns.
  • temporal patterns of expression can be provided by, for example, conditional recombination systems or prokaryotic transcriptional regulatory sequences.
  • the "transgenic non-human animals" of the invention are produced by introducing transgenes into the germline of the non-human animal.
  • Embryonal target cells at various developmental stages can be used to introduce transgenes. Different methods are used depending on the stage of development of the embryonal target cell.
  • the specific line(s) of any animal used to practice this invention are selected for general good health, good embryo yields, good pronuclear visibility in the embryo, and good reproductive fitness.
  • the haplotype is a significant factor. For example, when transgenic mice are to be produced, strains such as C57BL/6 or FVB lines are often used (Jackson Laboratory, Bar Harbor, Me.).
  • Preferred strains are those with H-2b , H-2 d or H-24 haplotypes such as C57BL/6 or DBA/1.
  • the line(s) used to practice this invention may themselves be transgenics, and/or may be knockouts (i.e., obtained from animals which have one or more genes partially or (completely suppressed) .
  • gene targeting which is a method of using homologous recombination to modify an animal's genome, can be used to introduce changes into cultured embryonic stem cells. By targeting an LTM gene of interest in ES cells, these changes can be introduced into the germlines of animals to generate chimeras.
  • the gene targeting procedure is accomplished by introducing into tissue culture cells a DNA targeting construct that includes a segment homologous to a target LTM locus, and which also includes an intended sequence modification to the LTM genomic sequence (e.g., insertion, deletion, point mutation). The treated cells are then screened for accurate targeting to identify and isolate those which have been properly targeted.
  • a DNA targeting construct that includes a segment homologous to a target LTM locus, and which also includes an intended sequence modification to the LTM genomic sequence (e.g., insertion, deletion, point mutation).
  • Gene targeting in embryonic stem cells is in fact a scheme contemplated by the present invention as a means for disrupting an LTM gene function through the use of a targeting transgene construct designed to undergo homologous recombination with one or more LTM genomic sequences.
  • the targeting constrict can be arranged so that, upon recombination with an element of an LTM gene, a positive selection marker is inserted into (or replaces) coding sequences of the targeted gene.
  • the inserted sequence functionally disrupts the LTM gene, while also providing a positive selection trait.
  • Exemplary LTM targeting constructs are described in more detail below.
  • Fig. 1 Time-course Northern blot analysis of zif268 and c-fos following IA training. Increase in zif268, but not c-fos mRNA is evident in all animals at 9 and 20 hr after training. These results showed that zif268 is induced in the hippocampus by IA. They also reveal when the induction of an IEG is detectable in the hippocampus following IA, namely at 9 and 20 hrs. On the basis of these data, as described below, we decided to perform our first array hybridization analysis using the RNA obtained from animals 9 hr after training.
  • FIG. 3 Broad scale expression profiling with cDNA expression arrays. Side by side hybridizations with cDNA probes prepared from two different RNA populations allow the simultaneous comparison of the expression levels of all the cDNAs on the array, (from Clontech user manual). Arrays can contain, in principle, all the cloned DNA sequences. We begun our analysis using a relatively small array, the AtlasTM rat cDNA expression array from Clontech (PaloAlto, Ca), which was the most complete commercially available array at that time.
  • This array contained 588 genes isolated from rat brain and coding for a variety of molecules involved in the pathways that regulate brain function, such as signaling molecules, receptors, signal transduction proteins, extracellular proteins, structural molecules, molecules involved in synaptic transmission, and molecules involved in neural pathologies, including Alzheimer disease.
  • the DNAs were fixed on a positively charged nylon membrane.
  • Figure 4 Examples of changes in gene array hybridizations reflecting differential expression of mRNAs following IA training. Hippocampi of control rats are compared to hippocampi of rats trained and sacrificed 9 hr later. Note that on these arrays each sequence is spotted in duplicate. The array hybridization is a powerful method of screening. However, to prove that the transcripts identified with this technique indeed change their expression following IA learning, a Northern blot hybridization analysis of each identified sequence was necessary. The Northern blot, and not the array hybridization, ultimately defines whether a gene changes its expression after learning, therefore all the necessary controls were carried out with the Northern blot hybridization and not with array hybridization.
  • the delayed and prolonged gene response during memory consolidation makes feasible detection of changes after the Morris water maze.
  • the Morris water maze is a very reliable and widely used assay of hippocampal function. Rats with damage to any part of the hippocampal system including the hippocampal formation itself, the entorhinal cortex and fornix show severe deficits on this task (Hannenson, 1998; Sutherland, 1989).
  • the water maze apparatus consists of a large, circular pool filled with water made opaque by the addition of white tempura paint. An escape platform is hidden just below the surface of the water, and the rat is required to swim to the hidden platform from different starting points around the edge of the pool. Normal rats can acquire this task quickly by using and remembering configurations of extramaze cues to navigate directly to the platform. Learning is reflected in shorter latencies to escape and decreases in the length of the path that the rat swims in order to reach the platform.
  • transcripts including genes and expressed sequence tag (ESTs)
  • ESTs expressed sequence tag
  • the array hybridization screening for genes differentially expressed following IA training will be carried on arrays purchased from Research Genetics (Huntsville, AL). This company has recently released the most complete rat DNA array commercially available, the rat GeneF ⁇ lters microarrays. These arrays contain over 5,000 spots, representing approximately 1,700 named rat genes as well as many rat ESTs that are considered similar to named genes in other organisms. Each spot on the membrane contains approximately 0.5 ng of insert DNA from a cDNA clone containing the 3' end of a gene. The insert cDNA has been denatured and UV-cross-linked to the positively charged membrane.

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Abstract

L'invention concerne la découverte de gènes qui sont régulés positivement ou négativement dans des comportements inhibiteurs d'évitement, p. ex. mémoire à long terme, ces gènes étant supposés intervenir dans la consolidation de la mémoire. En particulier, il a été découvert que la consolidation de la mémoire implique la régulation de l'expression de gènes tels que zif268 (EGR1), le facteur de croissance analogue à l'insuline (IGF-1), le récepteur 1 du glutamate (Glur1), le récepteur 2 du glutamate (GluR2), c/EBPβ et VGF. A des fins de commodité de lecture, ces gènes sont appelés collectivement 'gènes LTM ', et leurs protéines codées ' protéines LTM'.
EP01924598A 2000-03-31 2001-04-02 Procedes et compositions pour reguler la consolidation de la memoire Withdrawn EP1331930A2 (fr)

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US19361400P 2000-03-31 2000-03-31
US193614P 2000-03-31
PCT/US2001/010661 WO2001074298A2 (fr) 2000-03-31 2001-04-02 Procedes et compositions pour reguler la consolidation de la memoire

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US6689557B1 (en) * 1994-10-07 2004-02-10 Cold Spring Harbor Laboratory Method of identifying a substance that affects long term memory and CREM/CREB/ATF-1 subfamily members
WO2002052042A2 (fr) * 2000-12-22 2002-07-04 Medical Research Council Procede d'analyse de composes
WO2003060516A1 (fr) * 2002-01-10 2003-07-24 Takeda Chemical Industries, Ltd. Methode de criblage
AUPS255202A0 (en) * 2002-05-27 2002-06-13 Monash University Agents and methods for the treatment of disorders associated with motor neuron degeneration
US20060040338A1 (en) * 2004-08-18 2006-02-23 Odyssey Thera, Inc. Pharmacological profiling of drugs with cell-based assays
US20060094059A1 (en) * 2004-09-22 2006-05-04 Odyssey Thera, Inc. Methods for identifying new drug leads and new therapeutic uses for known drugs

Citations (2)

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Publication number Priority date Publication date Assignee Title
WO1997046257A1 (fr) * 1996-06-03 1997-12-11 The Trustees Of Columbia University In The City Of New York Procede permettant d'ameliorer la memoire a long terme chez un sujet et utilisations de ce procede
WO2001068137A2 (fr) * 2000-03-14 2001-09-20 Brown University Research Foundation Procedes et preparations pour la regulation de la consolidation de la memoire

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
WO1997046257A1 (fr) * 1996-06-03 1997-12-11 The Trustees Of Columbia University In The City Of New York Procede permettant d'ameliorer la memoire a long terme chez un sujet et utilisations de ce procede
WO2001068137A2 (fr) * 2000-03-14 2001-09-20 Brown University Research Foundation Procedes et preparations pour la regulation de la consolidation de la memoire

Non-Patent Citations (3)

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Title
DATABASE BIOSIS [Online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; February 1999 (1999-02) ALEMAN ANDRE ET AL: "Insulin-like growth factor-I and cognitive function in healthy older men." Database accession no. PREV199900133267 XP002241280 & JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM, vol. 84, no. 2, February 1999 (1999-02), pages 471-475, ISSN: 0021-972X *
RICKARD, N. S. ET AL: "Blockade of metabotropic glutamate receptors prevents long - term memory consolidation" BRAIN RESEARCH BULLETIN (1995), 36(4), 355-9 , XP002241279 *
See also references of WO0174298A2 *

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EP1331930A4 (fr) 2003-08-06
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JP2004508288A (ja) 2004-03-18
WO2001074298A2 (fr) 2001-10-11

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