EP1161525A2 - Methods and compositions for regulating memory consolidation - Google Patents

Abstract

The present invention provides methods for identifying genes involved in memory consolidation, and for identifying agents which effect memory consolidation in a mammal. These methods preferably use (1) non-human mammals which have been manipulated to create at least partial disruption of fornix-mediated afferant signalling to the hippocampus, said disruption affecting memory consolidation or (2) non-lesioned mammals which have been subjected to a learning and memory test to provide a learned behavior in the mammal.

Description

Methods and Compositions for Regulating Memory Consolidation

Government Support

This invention was partially funded by grants from certain government agencies; the government has certain rights to the invention.

Background of the Invention

Since the celebrated case of patient H.M., first described over 40 years ago, it has been appreciated that structures in the human temporal lobes play a special role in memory (Scoville et al. (1957) J. Neurol. Neurosurg. Psvchiat, 20:11-21). The syndrome caused by bilateral lesions of the temporal lobes is characterized by a profound anterograde amnesia, an inability to store new information into an accessible long-term memory. Recall of previously stored information, however, is relatively intact. Subsequent research on brain-damaged humans and animals has focused attention on several interconnected limbic structures that, together, have been considered as components of a "memory system" in the mammalian brain. See, e.g., Squire, L.R. Memory and Brain (Oxford, New York, 1987). Although the different components may be specialized to regulate different types of information storage, together the system is crucial for what have been called "declarative" memories. A loss of the ability to form this type of memory is a consequence of many neurological disorders that affect the temporal lobes, and it is also often a consequence of normal aging (Milner et al. (1998) Neuron 20:445). The mechanisms by which this system controls information storage are unknown.

The past several decades have also witnessed steady progress in understanding the cellular biology of information storage in the nervous system. In a series of classic studies beginning in the 1960s, it was shown that inhibition of mRNA and protein synthesis during a critical time window (during and shortly after training) could disrupt the formation of long-term memory (Flexner et al. (1963) Science 141:57; Barondes, Sl l Protein synthesis dependent and protein synthesis independent memory storage processes. In Short-Term Memory (eds. Deutsch, D.&D., J. A.) 379-390 (Academic, New York, 1975); Davis et al. (1984) Psvchol. Bull. 96:518; Castellucci et al. (1989) L Neurobiol. 20: 1-9). Initial learning and recall of previously stored information were not affected by the: transient blockade of protein synthesis. Thus it was hypothesized that new gene expression is required for the conversion or consolidation of a short-term modification of the brain into a long-term memory. More recently, it has been shown that formation of long term memory depends specifically on activation of gene expression regulated by members of the cAMP response element binding protein (CREB) transcription factor family (Tully et al. (1994) Cell 79:35; Bartsch et al. (1995) Cell 83:979; Guzowski et al. (1997) PNAS 94:2693; Silva et al. (1998) Ann. Rev. Neurosci. 21 :127). A necessary step for transcriptional activation is phosphorylation of CREB at Ser-133, which occurs in response to activation of a number of different intracellular second messenger pathways (Impey et al. (1998) Neuron 21:869; Silva et al. supra). However, crucial questions remain about how, when and where CREB- dependent gene expression is regulated in the brain during long-term memory formation.

The apparently similar behavioral consequences of temporal lobe lesions and protein synthesis inhibitors have led to the suggestion that the temporal lobe memory system is involved in the direct regulation of the neuronal gene expression that is required for establishment of long-term memory. Here we report a test or this hypothesis. Rats received inhibitory avoidance training, which produces long-term memory in a single trial, and various brain regions were assayed for time-dependent changes in CREB phosphorylation at Ser-133. This approach revealed a circumscribed and persistent CREB phosphorylation in the neurons of the hippocampal formation following training. We then investigated whether this CREB response was altered by limbic lesions that produce amnesia on this task. Lesions of the fornix, which produce a marked impairment in long-term memory, completely prevent the CREB phosphorylation following training. Our data therefore suggest a possible molecular basis for the amnesia That results from damage to the temporal lobe memory system.

Summary of the Invention

One aspect of the present invention provides methods for identifying agents which enhance memory consolidation in a mammal.

In certain embodiments, the assays utilize a non-human mammal which has been manipulated to create at least partial disruption of fornix-mediated afferant signalling to the hippocampus, said disruption affecting memory consolidation. For instance the animal can be generated by by mechanical or chemical disruption of at least a portion of the fornix. In certain preferred embodiments, the fornix lesion is generated by selective disruption of one or more neuronal types, e.g., generated by selective disruption of one or more the neurons selected from the group consisting of fornix cholinergic neurons, fornix GABAergic neurons and fornix serotonergic neurons. In certain embodiments, the method includes a step of conditioning the mammal with a learning or memory regimen which results in learned behavior in the mammal in the absence of the fornix lesion. A test agent which is to be assessed for effects on memory consolidation is administered to the animal, and the effect of the test agent on the the learned behavior of the mammal is assessed. An increase in learned behavior, relative to the absence of the test agent, indicates that the test agent enhances memory consolidation. For instance, if the mammal retains the learned behavior after at least about 24 hours, the agent can is one which enhances memory consolidation.

In other embodiments, the effect of the test agent on the fornix-lesioned animal is tested by determining the extent of phosphorylation of CREB in the hippocampus of the mammal and comparing that to the extent of phosphorylation of CREB in an untrained control mammal. If the extent of phosphorylation of CREB in the test mammal is greater than the extent of phosphorylation of CREB in the control mammal, the agent promotes memory consolidation. In certain preferred embodiments, the animal is a rodent, such as a mouse or rat.

The animal may also be a transgenic animal.

In certain preferred embodiments, the test agent is a organic molecule having a molecular weight less than 2500 amu.

In certain preferred embodiments, the method is carried out for a plurality of different test agents, e.g., a library, e.g., at least 10, 100 or 1000 different test agents.

Another aspect of the present invention relates to pharmaceutical preparations including one or more compounds identified by the subject methods, e.g., formulated in a pharmaceutically acceptable excipient.

Another aspect of the present invention relates to a method for the identification of mammalian genes involved in memory consolidation. This method generall involves comparing the level of expression of genes from a control animal, e.g., one which has undergone memory consolidation or is untrained, with the level of expression of genes from a test animal, e.g., and animal having at least partial disruption of fornix-mediated afferant signalling to the hippocampus which disrupts memory consolidation. Genes that are up-or downregulated in the test animal relative to the the control animal are candidate genes for involvment in memory consolidation, e.g., "LTM genes".

For example, the subject method can be practiced by differential cloning teachniques. For example, a first library of nucleic acid probes representative of genes expressed in animals having undergone memory consolidation can be compared with a second library of nucleic acid probes representative of genes expressed in animals having a fornix lesion that affects memory consolidation. Genes that are that up-or downregulated in the first library of nucleic acids relative to the second library of nucleic acids are identified. In preferred embodiments, the nucleic acid libraries are derived from hippocampal tissue.

In certain embodiments, the assay can include a step of detecting the level of activity of a product encoded by a gene that is identified as being up- or downregulated in the first library of nucleic acids.

Another aspect of the invention provides a method for identifying an agent which modulates memory consolidation by targeting the LTM genes and gene products identified by the methods described herein. In general, the method involves providing a reaction system for detecting the activity of a product encoded by an LTM gene. The system is contacted with a test compound, and the ability of the test compound to alter the activity of the gene product is determined. Another aspect of the invention provides a method for identifying an agent which modulates memory consolidation by altering the level of expression of an LTM gene. Generally, the method involves providing a reaction system for detecting the level of expression of an LTM gene, contacting the system with with a test compound, and determining if the test compound alters the level of expression of the gene. Another aspect of the invention provides a method for enhancing memory consolidation in an animal, or otherwise enhancing the functional performance of CNS neurons, by administering a pharmaceutical preparation of a drug identified by the assays disclosed herein. For example, the treatment can be for augmenting learning and memory. In certain embodiments, the method includes administering, conjointly with the pharmaceutical preparation, one or more of a neuronal growth factor, a neuronal survival factor, and a neuronal tropic factor.

In certain embodiments, the method includes administering, conjointly with the pharmaceutical preparation, an agent that activates CREB-dependent transcription in an amount sufficient to produce a memory enhancing effect. For instance, the CREB activating agent can be a cAMP elevating agent, e.g., an adenylate cyclase activator a cAMP analog, or a cAMP phosphodiesterase inhibitor.

Another aspect of the present invention provides a method for assessing a patient for learning and/or memory functional performance by detecting the level of CREB phosphorylation in the patient's hippocampus. For instance, the level of CREB phosphorylation in the patient's hippocampus is detected by non-invasive spectroscopy, such as Magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS), cerebral computed tomography (CCT), in vivo nmr (e.g., 31p NMR), or positron emission tomography (PET) imaging.

Another aspect of the present invention provides a method for assessing a patient for learning and/or memory functional performance by detecting the expression of one or more LTM genes identified according to the present methods.

Still another aspect of the invention provides a method of identifying an agent which modulates memory consolidation in a mammal by recapitulating an aspect of fornix signalling. In general, the method involved

(i) providing a non-human mammal which has been manipulated to create at least partial disruption of fornix-mediated afferant signalling to the hippocampus, said disruption affecting memory consolidation, (ii) conditioning the mammal with a learning or memory regimen which results in learned behavior in the mammal in the absence of the fornix lesion;

(iii) ascertaining the effect of one or neurotransmitters, or agonist or antagonist thereof, on the the learned behavior of the mammal. A change in learned behavior, relative to the absence of the neurotransmitter(s), indicates that the neurotransmitter effects memory consolidation.

In certain preferred embodiments, the method is carried out by conjointly administering two or more neurotransmitters, or agonists or antagonists thereof.

Yet another aspect provides a method for enhancing memory consolidation in an animal, comprising administering to the animal one or more neurotransmitters, or agonists or antagonists thereof, produced by fornix-mediated afferant signalling to the hippocampus affecting memory consolidation. In preferred embodiments, two or more neurotransmitters, or agonists or antagonists thereof, produced by fornix-mediated afferant signalling to the hippocampus affecting memory consolidation are administered. In certain embodiments, at least one of the neurotransmitters is an agonist of a neurotransmiter which enhances memory consolidation. In certain embodiments, at least one of the neurotransmitters is an antagonist of a neurotransmiter which inhibits memory consolidation.

Another aspect of the invention provides a pharmaceutical preparation comprising two or more neurotransmitters, or agonists or antagonists thereof, produced by fornix-mediated afferant signalling to the hippocampus, which neurotransmitters are provided in an amount sufficient to affect memory consolidation in a mammal.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are described in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucieotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Patent No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); fmmobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immuno chemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I- IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embiyo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

Brief Description of the Drawings

Figure 1. Lesions of the fornix result in an impairment on the inhibitory avoidance task, (a) Representative fornix lesion (asterisk), (b) Rats with fornix lesions performed at sham-control levels on the acquisition trial, and on retention testing when conducted at 0 and 6 hr after the training trial. By 24 hr, animals with fornix lesions exhibited a severe impairment as compared to control rats (fornix lesion, n = 8; sham- control, n = 8, p < 0.003).

Figure 2. A sustained increase in hippocampal CREB phosphorylation occurs following inhibitory avoidance training in normal rats but not in rats with fornix lesions. Western blot analyses of hippocampal extracts from unoperated and fornix-lesion rats were carried out with anti-PCREB and anti-CREB. (a) Examples of PCREB and CREB western blot immuno staining of hippocampal extracts from unoperated and fornix-lesion animals. Three examples are shown for 3 conditions: (1) animals that entered the training apparatus but received no shock, (2) animals that entered the apparatus, received the training shock and were immediately sacrificed ("0 hr"), and (3) animals that entered the apparatus, received the training shock and were sacrificed 6 hrs later C6 hr"). (b) Densitometric analysis of PCREB western blot immunostaining of unoperated and fornix-lesion hippocampi taken from animals at vaiious timepoints after training, pd from "no shock" controls. Unoperated animals showeda significant increase in PCREB at 0 hr (n = 8, p < 0.05), 3 hr (n 4, p < 0.05) and 6 hr (n = 8, p < 0-05) after training compared to "no shock" controls. In contrast, animals with fornix lesions failed to show any significant increase in PCREB after training. Data are expressed as mean percentage ± SEM of the "no shock" unoperated control mean values, (c) Densitometric analysis of CREB western blot immunostaining, performed on the same samples described in (b). There are no significant differences in the total amount of CREB in hippocampi of unoperated and fornix lesion animals at 0, 3 or 6 hr after training. Data are expressed as mean percentage ± SEM of the "no shock" unoperated control mean values (n = 4 per timepoint). (d) Densitometdc analysis of PCREB western blot immunostaining of hippocampi from unoperated rats sacrificed at 0, 3, 6, and 9 hr after exposure to the training apparatus without receiving footshock. No significant changes in PCREB levels were found at any timepoint. Data are expressed as mean percentage ± SEM of the "no shock" unoperated control mean value (0 hr, n = 8; 3 hr, n = 4; 6 hr, n = 8; 9 hr, n = 4).

Figure 3. CREB phosphorylation after inhibitory avoidance learning is mostly induced in CA1 and dentate gyrus. Examples of immunohistochemical staining using anti-PCREB in unoperated (a and b) and fornix lesion (c and d) rat brain slices before (no shock) and immediately after footshock training (0 hr). 40 m sections (approx. 3.80 mm posterior to Bregma) magnified at lOx are shown. CA1 and dentate gyrus (DG) subregions are indicated.

Detailed Description of the Invention

I. Overview

Behavioral research has found that the human mind consolidates memory at certain key time intervals. The initial phase of memory consolidation occurs in the first few minutes after we are exposed to a new idea or learning experience. The next phase occurs during our sleep that night. 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.

It has been known for several decades that the formation of long-term memory requires gene expression. The prevailing hypothesis for the formation of long-term memory (LTM) is that 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).

In animal models of learning and memory, the requirement for de novo protein synthesis around the time of training has long been a definitive property of long-term memory that separates it from other types of memory retention. Thus, the storage of long-term memory is associated with cellular program(s) of gene expression, altered protein synthesis, and the growth of new synaptic connections. For instance, recent work suggests that this property of memory formation may have a specific molecular underpinning that involves cAMP-responsive transcription and that is mediated through the cAMP responsive element binding protein (CREB) family of transcription factors.

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 by protein kinase A, 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. In aplysia, for example, CREB activation has been interfered with by microinjection of CRE containing oligonucleotides into cultured neurons. In drosophila, CREB function has been disrupted using a reverse genetic approach. Thus, LTM has been specifically blocked by the induced expression of a CREB repressor isoform, and enhanced by the induced expression of an activator isoform. In mouse, 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 disrupted. On the whole, the data suggest that encoding of long term memories involve highly conserved molecular mechanisms.

Animals with lesions of the medial temporal lobes and related thalamic structures show a profound disruption of memory consolidation, but the cause of the amnesia is unknown. Here we present evidence indicating that one form of lesion-induced amnesia is associated with abnormal regulation of gene expression in specific subregions of the hippocampus. Inhibitory avoidance training produces a rapid and persistent increase in the phosphorylation of CREB, which is a necessary step in the regulation of CRE- mediated gene expression required for memory consolidation. The change in CREB phosphorylation is largely confined to hippocampal fields CA1 and dentate gyrus, and lasts at least 6 hours after training. 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 amnesic animals also fail to exhibit any increase in hippocampal CREB phosphorylation after training. Our results suggest that hippocampal inputs passing through the fornix regulate consolidation of this form of memory via regulation of CREB-mediated gene expression in hippocampal neurons.

One aspect of the present invention concerns assays and reagents for identifying other genes (herein "LTM genes") and gene products (herein "LTM proteins") which have roles in memory formation, and in particular is directed to mechanistic models pertaining to fornix- and hippocampal-mediated mechanisms that underlie LTM. In general, the method involves comparing the level of expression of genes from a control animal, characterized by having undergone memory consolidation or being untrained, with the level of expression of genes from a test animal, characterized by having at least partial disruption of fornix-mediated afferant signalling to the hippocampus so as to inhibit memory consolidation. Genes that are up-or downregulated in the test animal relative to the the control animal are candidate genes for involvment in memory consolidation.

Another aspect of the present invention relates to the use of such genes and their products for carrying out assays designed to identify agents which, by modulating the function of certain LTM genes, can be used to modify long term memory consolidation in animals. As described in further detail below, 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. The present invention also relates to a method of identifying an agent which modulates (enhances or inhibits) expression of an LTM gene (one or more) activated in the brain of a mammal during memory consolidation. In one embodiment, the agent to be assessed is administered to a mammal and the level of expression of an LTM gene in the hippocampus is assessed. In other embodiments, the agent to be assessed is administered to a mammal having a lesion in the fornix that disrupts memory consolidation, and the mammal is subjected to a learning and/or memory test to provide a learned behavior in the mammal. The pattern and amount of gene expression in the brain of the lesion mammal is determined and compared to the pattern and amount of gene expression in the brain of a control mammal. Agents which recapitulate, in the lesioned animal, at least a portion of the pattern of gene expression observed in the control animal are candidate agent for regulated memory consolidation.

The present invention also provides a method for identifying an agent which enhances memory consolidation utilizing an animal which has been manipulated to create at least partial disruption of fornix-mediated afferant signalling to the hippocampus, the disruption affecting memory consolidation. 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, wherein an increase in learned behavior, relative to the absence of the test agent, indicates that the test agent enhances memory consolidation.

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. As a result, 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.

Yet another aspect of the invention relates to the use of compounds which recapitulate the action of neurotransmitters produced by the fornix-mediated afferant signalling to the hippocampus that effects memory consolidation. For example, the subject invention provides a method for enhancing memory consolidation in an animal with a treatment regimen including administering to the animal one or more neurotransmitters, or agonists or antagonists thereof, produced by fornix-mediated afferant signalling to the hippocampus affecting memory consolidation. In certain preferred embodiments, the method involves the conjoint administration of at least two different agents which mimic the action of neurotransmitters produced by fornix neurons.

II. Definitions For convenience, the meaning of certain terms and phrases employed in the specification, examples, and appended claims are provided below.

"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. The "CREB" family of proteins (sometimes referred to as the ATF family) is best known for the three members that can mediate cAMP -responsive transcription: CREB itself, CREM and ATF-1 (DeGroot et al. (1993) Mol Endocrinol 7:145-153). These basic-region, leucine-zipper proteins bind to DNA sequences, called cAMP response element (CRE) sites, which are often found in the upstream regulatory regions of genes whose synthesis is cAMP responsive. Molecular analysis has shown that CRE sites, and their interaction with CREB family members, are necessary for cAMP responsiveness. After the catalytic subunit of PKA translocates to the nucleus, it can directly phosphorylate the serine residue at position 133 on CREB, thus activating the protein and directly linking the cAMP transduction pathway to the induction of new gene expression (Backsai et al. (1993) Science 260: 222-226; and Hagiwara et al. (1993) Mol Cell Biol 13:4852-4859)

As is well known, 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. Moreover, 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.

As used herein, 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. When an equivalent position in the compared sequences is occupied by the same base or amino acid, then 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. 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. In one embodiment, 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. The term "interact" as used herein is meant to include detectable interactions between molecules, such as can be detected using, for example, a two hybrid assay. The term interact is also meant to include "binding" interactions between molecules. Interactions may be protein-protein or protein-nucleic acid in nature. The term "isolated" as used herein with respect to nucleic acids, such as DNA or

RNA, refers to molecules separated from other DNAs or RNAs, respectively, that are present in the natural source of the macromolecule. For example, 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. The term isolated as used herein 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. Moreover, an "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. The term "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.

The term "modulation" as used herein refers to both upregulation, i.e., stimulation, and downregulation, i.e. suppression, of a response.

The "non-human animals" of the invention 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. The term "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. The term "tissue-specific chimeric animal" indicates that one of the recombinant genes is present and/or expressed or disrupted in some tissues but not others.

As used herein, the term "nucleic acid" refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). 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.

As used herein, the term "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. The term encompasses "tissue specific" promoters, i.e. promoters, which effect expression of the selected DNA sequence only in specific cells (e.g. cells of a specific tissue). The term also covers 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).

The terms "protein", "polypeptide" and "peptide" are used interchangably herein when referring to a gene product. The term "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. Moreover, the 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. In preferred embodiments, 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. It will also be understood that 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.

As used herein, 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", as used herein, 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. As used herein, the term "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. 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. However, transgenic animals in which the recombinant gene is silent are also contemplated, as for example, the FLP or CRE recombinase dependent constructs described below. Moreover, "transgenic animal" also includes those recombinant animals in which gene disruption is caused by human intervention, including both recombination and antisense techniques.

As used herein, the term "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". In general, 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. In the present specification, "plasmid" and "vector" are used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forts of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.

III. Exemplary Embodiments

A. Generation of Animal Models

We have discovered that long-term memory consolidation includes fornix- mediated gene expression in the hippocampus. One aspect of the present invention provides an animal model for studying fornix-mediated memory consolidation. For instance, the subject animals can be used for drug screening, e.g., to identify compounds which enhance memory consolidation, as well as identifying hippocampal genes which are up- or down-regulated in memory consolidation as a result of fornix-mediated afferant signalling to the hippocampus. As described in further detail below, inhibitory avoidance training produces a rapid and persistent increase in the phosphorylation of CREB (cyclic AMP response element binding protein), which is a necessary step in the regulation of CRE-mediated gene expression required for memory consolidation. The change in CREB phosphorylation is largely confined to hippocampal fields CA1 and dentate gyrus, and lasts at least 6 hours after training. 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 amnesic animals also fail to exhibit any increase in hippocampal CREB phosphorylation after training. Our results suggest that hippocampal inputs passing through the fornix regulate consolidation of this form of memory via regulation of CREB-mediated gene expression in hippocampal neurons.

In the methods of the present invention, 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. For example, 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.

To further illustrate, fimbrio-fornix (rodents) and fornix (primates) lesions can be created by stereotatic ablation. In particular, 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. In general, the fornix transection utilized in the subject method will not disconnect the parahippocampal region from the neocortex. In those embodiments, the fornix transection can be carried out so as not to disrupt functions that are 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.

In one embodiment, the animal can be a rat. Briefly, the animals are anesthetized, e.g., with intraperitoneal injections of a ketamine-xylazine mixture and positioned in a 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 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.

In another exemplary embodiment, the animal can be a monkey. The animals can be anesthetized, e.g., with isoflurane (1.5-2.0%). Following pretreatment with 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. Briefly, 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. At the level of the foramen of Monro, 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.

In still other illustrative embodiments, 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. In certain preferred embodiments, 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. For instance, 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 methylenedioxymetharnphetamine (MDMA), and 5,7-dihydroxytryptamine (5,7-DHT). The afferant fornix signals to the hippocampus due to cholinergic neurons can be ablated by atropine blockade. Another means for ablation of the cholinergic neurons is the use of 192IgG-saporin (192IgG- sap), e.g., intraventricularly injection into the fornix and hippocampus. In other embodiments, 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-methyl-D-aspartate (NMD A), quinolinic acid, and methylazoxymethanol. In preferred embodiments, the animal is a non-human mammal, such as a dog, cat, horse, cow, pig, sheep, goat, chicken, monkey, ape, rat, rabbit, etc. In certain preferred embodiments, the animal is a non-human primate. In other preferred embodiment, the animal is a rodent.

There are a variety of test for cognitive function, especially learning and memory testing, which can be carried our using the lesioned and normal animals. 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 passive avoidance test utilizes an apparatus that consists of a lit chamber that can be separated from a dark chamber by a sliding door. At training, 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. Upon entry into the dark chamber, 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 passive avoidance procedures (for review, see Rush (1988) Behav Neural Biol 50:255).

An exemplary maze testing embodiment is the water maze memory test. In general, 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. During the experiment, 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 preceedes 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. In one embodiment, 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. Fruit Loop) in 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. When an individual hole is baited, a small piece of Fruit 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.

B. Use of Animal Models for Drug Testing One use for the fornix-lesioned animals of the present invention is for identifying compounds, e.g, from amongst various "test agents", which are able to enhance or inhibit memory consolidation. In general, 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. In the methods of the present invention, 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.

As used herein, a "control mammal" can be an untreated lesion mammal (i.e., a lesion animal receiving no agent to be assessed), a trained control mammal (i.e., a mammal that undergoes training to demonstrate a learned behavior without any lesion) and/or an untrained control mammal (i.e., a mammal with or without a lesion, that receives no training to demonstrate a learned behavior).

The agent to be assessed in the methods of the present invention can be administered to the mammal either before or after the mammal is subjected to a learning and memory test to provide a learned behavior in the mammal. Any agent can be assessed in the methods of the present invention. Exemplary test agents 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 one emboidment, the animal is is treated with a combination of agents which are selected on the basis of recapitulating the activity of neurotransmitters and hormones (e.g., acetylcholine, doparnine, serotonin, norepinephrine, cortisol, etc.) which are required for fornix-mediated LTM. In addition to the neurotransmitters and hormones themselves, the assay can be carried out using agonists and antagonists of such neurotransmitter receptors (e.g., glutarnate, acetylcholine, doparnine, serotonin, norepinephrine, cortisol receptors), second messenger modulatory agents (e.g., cyclic AMP analogues, phorbol esters, phophodiesterase inhibitors) and agents affecting CREB phophorylation (e.g., cyclic-AMP-dependent protein kinase, MAP kinase, calcium- calmodulin-dependent protein kinase).

Determining the ability of the mammal to retain the learned behavior after completion of the learning phase can be performed in a variety of ways. For example, the amount of CREB phosphorylation can be determined or a learning and memory test can be preformed.

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. Compounds which are identified as active in the subject assay can be subjected to further testing, as well as medicinal chemistry and structure-activity relationship studies in order to optimize a drug candidate. Candidate drugs may be formulated in a pharmaceutically acceptable excipient, and administered to animals for further testing and/or for treatment.

In other embodiments, the subject method can be used to identify an agent which promotes memory consolidation by detecting the ability of the test agent to regulate CREB phosphorylation in the hippocampus. In general, this embodiment of the assay includes administering the agent to be assessed to a mammal that has been subjected to a learning and memory test to provide a learned behavior in the mammal. The extent of phosphorylation of CREB in the hippocampus of the mammal is assessed and compared to the extent of phosphorylation of CREB in the hippocampus of an untrained control mammal. If the extent of phosphorylation of CREB in the trained mammal is greater than the extent of phosphorylation of CREB in the control mammal, the agent promotes memory consolidation. In certain preferred embodiments, the trained mammal has been manipulated to create at least partial disruption of fornix-mediated afferant signalling to the hippocampus, said disruption affecting memory consolidation

In still other embodiments, the present invention provides a method of identifying an agent which enhances or inhibits expression of one or more LTM genes involved in memory consolidation. To illustrate, the agent to be assessed can be administered to a mammal and the level of expression of an LTM gene in the hippocampus is assessed. In other embodiments, the agent to be assessed is administered to a mammal having a lesion in the fornix that disrupts memory consolidation, and the mammal is subjected to a learning and/or memory test to provide a learned behavior in the mammal. The pattern and amount of gene expression in the brain of the lesion mammal is determined and compared to the pattern and amount of gene expression in the brain of a control mammal. Agents which recapitulate, in the lesioned animal, at least a portion of the pattern of gene expression observed in the control animal are candidate agent for regulated memory consolidation.

C. Use of Animal Models for Genomics

In one embodiment, the subject method can be used for the identification of mammalian genes, termed herein "long-term memory genes" or "LTM" genes, involved in fornix-mediated memory consolidation. In general, the method comprises comparing the level of expression of genes from a "control animal", e.g., one which has undergone memory consolidation or is untrained, with the level of expression of genes from a "test animal", e.g., one which is characterized by having at least partial disruption of fornix- mediated afferant signalling to the hippocampus affecting memory consolidation. Genes that are up- or down-regulated in the test animal relative to the the control animal are candidate genes for involvment in memory consolidation.

In one embodiment, LTM genes may be identified by a method generally including the steps of: (i) generating a first library of nucleic acid probes representative of genes expressed in animals having undergone memory consolidation; (ii) generating a second library of nucleic acid probes representative of genes expressed in animals having at least partial disruption of fornix-mediated afferant signalling to the hippocampus, said disruption affecting memory consolidation; (iii) identifying genes that up-or downregulated in the first library of nucleic acids relative to the second library of nucleic acids. In certain embodiments, the first and second nucleic acid libraries are derived from hippocampal tissue. In certain embodiments, the subject method can also include a further step of detecting the level of activity of a product encoded by a gene that is identified as being up- or downregulated in the first library of nucleic acids.

A number of methods have been developed for the detection and isolation of genes which are activated or repressed in response to developmental, physiological, pharmacological, or other cued events. It is expected that such techniques may be readily adapted for use int he present invention.

One particular method is described in U.S. Patent 5,525,471 to Zeng, is subtractive hybridization. Subtractive hybridization is a particularly useful method for selectively cloning sequences present in one DNA or RNA population but absent in another. The selective cloning is accomplished by generating single stranded complementary DNA libraries from both control cells/tissue (driver cDNA) and cell/tissue during or after a specific change or response being studied (tester cDNA). The two cDNA libraries are denatured and hybridized to each other resulting in duplex formation between the driver and tester cDNA strands. In this method, common sequences are removed and the remaining non-hybridized single-stranded DNA is enriched for sequences present in the experimental cell/tissue which is related to the particular change or event being studied.

Other exemplary differential cloning techniques are described in, for example,

Zeng et al., (1994) "Differential cDNA cloning by enzymatic degrading subtraction

(EDS)", Nucleic Acids Research. 22:4381; Hubank et al., (1994) "Identifying differences in mRNA expression by representational difference analysis of cDNA", Nucleic Acids Research. 22:5640; Suzuki et al, (1996) "Efficient isolation fo differentially expressed genes by means of a newly established method, 'EDS'", Nucleic Acids Research. 24:797; Milner, (1995) "A kinetic model for subtractive hybridization", Nucleic Acids Research. 23:176; Kunkel et al, (1996) "Specific cloning of DNA fragments absent from the DNA of a male patient with an X chromosome deletion", PNAS. 82:4778; Liang et al., (1993) "Distribution and cloning of eukaryotic mRNAs by means of differential display: refinements and optimization", Nucleic Acids Res. 21 :3269; and Supplement 34 (1996) "PCR-Based Substractive cDNA Cloning", Sections 5.9.1-5.11.1, Ausubel, F.M., et al. (1989) Current Protocols in Molecular Biology. Greene Publishing Associates and Wiley-Interscience, New York.

In certain instances, it will be desirable to utilize a normalized library, which are constructed in a manner that increases the relative frequency of occurrence of rare clones while decreasing simultaneously the relative frequency of the occurrence of abundant clones. For teaching regarding the production of normalized libraries, see, e.g., Soares et al. (Soares, M.B. et al., 1994, Proc. Natl. Acad. Sci. USA 91 :9228-9232, which is incorporated herein by reference in its entirety). Alternative normalization procedures based upon biotinylated nucleotides may also be utilized, and are described in greater detail below.

D. Nucleic Acids encoding LTM proteins

As described below, one aspect of the invention pertains to isolated nucleic acids comprising nucleotide sequences encoding a protein involved in memory consolidation, which proteins will be herein referred to as "LTM" proteins or polypeptides. The term 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. Still other preferred 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. For example, preferred nucleic acid molecules for use as probes/primer or antisense molecules (i.e. noncoding nucleic acid molecules) can comprise at least about 6, 12, 20, 30, 50, 100, 125, 150 or 200 base pairs in length, whereas 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. For example, 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. In addition, 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%o, 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.

It should also be possible to obtain nucleic acids encoding LTM polypeptides cloned by the subject method from genomic DNA from both adults and embryos. For example, a gene encoding an LTM protein can be cloned from either a cDNA or a genomic library in accordance with protocols described herein, as well as those generally known to persons skilled in the art. Examples of tissues and/or libraries suitable for isolation of the subject nucleic acids include breast, among others. A cDNA encoding an LTM protein can be obtained by isolating total mRNA from a cell, e.g. a vertebrate cell, a mammalian cell, or a human cell, including embryonic cells. Double stranded cDNAs can then be prepared from the total mRNA, and subsequently inserted into a suitable plasmid or bacteriophage vector using any one of a number of known techniques. The gene encoding an LTM protein can also be cloned using established polymerase chain reaction techniques in accordance with the nucleotide sequence information provided by the invention. The nucleic acid of the invention can be DNA or RNA or analogs thereof. (i) Expression Vectors.

This invention also provides expression vectors containing a nucleic acid encoding an LTM polypeptide, operably linked to at least one transcriptional regulatory sequence. "Operably linked" is intended to mean that the nucleotide sequence is linked to a regulatory sequence in a manner which allows expression of the nucleotide sequence. Regulatory sequences are art-recognized and are selected to direct expression of the subject LTM proteins. Accordingly, the term "transcriptional regulatory sequence" includes promoters, enhancers and other expression control elements. Such regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). In one embodiment, the expression vector includes a recombinant gene encoding a peptide having an agonistic activity of a subject LTM polypeptide, or alternatively, encoding a peptide which is an antagonistic form of the LTM protein. Such expression vectors can be used to transfect cells and thereby produce polypeptides, including fusion proteins, encoded by nucleic acids as described herein. Moreover, the gene constructs of the present invention can also be used as a part of a gene therapy protocol to deliver nucleic acids encoding either an agonistic or antagonistic form of one of the subject LTM proteins. Thus, another aspect of the invention features expression vectors for in vivo or in vitro transfection and expression of an LTM polypeptide in particular cell types so as to reconstitute the function of, or alternatively, abrogate the function of LTM-induced signaling in a tissue, e.g., in hippocampal tissue.

In addition to viral transfer methods, such as those illustrated above, non-viral methods can also be employed to cause expression of a subject LTM polypeptide in the tissue of an animal. Most nonviral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules. In preferred embodiments, non-viral targeting means of the present invention rely on endocytic pathways for the uptake of the subject LTM polypeptide gene by the targeted cell. Exemplary targeting means of this type include liposomal derived systems, poly- lysine conjugates, and artificial viral envelopes.

(ii) Probes and Primers

Moreover, the nucleotide sequences determined from the cloning of LTM genes from mammalian organisms will further allow for the generation of probes and primers designed for use in identifying and/or cloning LTM homologs in other cell types, e.g. from other tissues, as well as LTM homologs from other mammalian organisms. For instance, the present invention also provides a probe/primer comprising a substantially purified oligonucieotide, which oligonucieotide comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least approximately 12, preferably 25, more preferably 40, 50 or 75 consecutive nucleotides of sense or anti- sense sequence of an LTM gene or naturally occ ring mutants thereof.

Likewise, probes based on the subject LTM sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In preferred embodiments, the probe further comprises a label group attached thereto and able to be detected, e.g. the label group is a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.

As discussed in more detail below, such probes can also be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress an LTM protein, such as by measuring a level of an LTM-encoding nucleic acid in a sample of cells from a patient; e.g. detecting LTM mRNA levels or determining whether a genomic LTM gene has been mutated or deleted. Briefly, nucleotide probes can be generated from the subject LTM genes which facilitate histological screening of intact tissue and tissue samples for the presence (or absence) of LTM-encoding transcripts. Similar to the diagnostic uses of anti-LTM antibodies, the use of probes directed to LTM messages, or to genomic LTM sequences, can be used for both predictive and therapeutic evaluation of allelic mutations which might be manifest in, for example, a predisposition to memory disorders. Used in conjunction with immunoassays as described herein, the oligonucieotide probes can help facilitate the determination of the molecular basis for a disorder which may involve some abnormality associated with expression (or lack thereof) of an LTM protein. For instance, variation in polypeptide synthesis can be differentiated from a mutation in a coding sequence.

(iii) Antisense, Ribozyme and Triplex Techniques

Another aspect of the invention relates to the use of the isolated LTM nucleic acids in "antisense" therapy. As used herein, "antisense" therapy refers to administration or in situ generation of oligonucieotide molecules or their derivatives which specifically hybridize (e.g. bind) under cellular conditions, with the cellular mRNA and/or genomic DNA encoding one or more of the subject LTM proteins so as to inhibit expression of that protein, e.g. by inhibiting transcription and/or translation. The binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix. In general, "antisense" therapy refers to the range of techniques generally employed in the art, and includes any therapy which relies on specific binding to oligonucieotide sequences. An antisense construct of the present invention can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes an LTM protein. Alternatively, the antisense construct is an oligonucieotide probe which is generated ex vivo and which, when introduced into the cell causes inhibition of expression by hybridizing with the mRNA and/or genomic sequences of an LTM gene. Such oligonucieotide probes are preferably modified oligonucleotides which are resistant to endogenous nucleases, e.g. exonucleases and/or endonucleases, and are therefore stable in vivo. Exemplary nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Pat. Nos. 5,176,996; 5,264,564; and 5,256,775). Additionally, general approaches to constructing oligomers useful in antisense therapy have been reviewed, for example, by Van der Krol et al. (1988) Biotechniques 6:958-976; and Stein et al. (1988) Cancer Res 48:2659-2668. With respect to antisense DNA, oligodeoxyribonucleotides derived from the translation initiation site, e.g., between the - 10 and + 10 regions of the LTM nucleotide sequence of interest, are preferred.

Antisense approaches involve the design of oligonucleotides (either DNA or RNA) that are complementary to LTM mRNA. The antisense oligonucleotides will bind to the LTM mRNA transcripts and prevent translation. Absolute complementarity, although prefeπed, is not required. A sequence "complementary" to a portion of an RNA, as referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.

Oligonucleotides that are complementary to the 5' end of the message, e.g., the 5' untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation. However, sequences complementary to the 3' untranslated sequences of mRNAs have recently been shown to be effective at inhibiting translation of mRNAs as well. (Wagner, R. (1994) Nature 372:333). Therefore, oligonucleotides complementary to either the 5' or 3' untranslated, non-coding regions of an LTM gene could be used in an antisense approach to inhibit translation of endogenous LTM mRNA. Oligonucleotides complementary to the 5' untranslated region of the mRNA should include the complement of the AUG start codon. Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention. Whether designed to hybridize to the 5', 3' or coding region of LTM mRNA, antisense nucleic acids should be at least six nucleotides in length, and are preferably less that about 100 and more preferably less than about 50, 25, 17 or 10 nucleotides in length.

Regardless of the choice of target sequence, it is preferred that in vitro studies are first performed to quantitate the ability of the artisense oligonucieotide to quantitate the ability of the antisense oligonucieotide to inhibit gene expression. It is preferred that these studies utilize controls that distinguish between antisense gene inhibition and nonspecific biological effects of oligonucleotides. It is also preferred that these studies compare levels of the target RNA or protein with that of an internal control RNA or protein. Additionally, it is envisioned that results obtained using the antisense oligonucieotide are compared with those obtained using a control oligonucieotide. It is preferred that the control oligonucieotide is of approximately the same length as the test oligonucieotide and that the nucleotide sequence of the oligonucieotide differs from the antisense sequence no more than is necessary to prevent specific hybridization to the target sequence.

The oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded, the oligonucieotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The oligonucieotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad.

Sci. USA 84:648-652; PCT Publication No. WO 88/09810, published Dec. 15, 1988) or the blood-brain baπier (see, e.g., PCT Publication No. WO 89/10134, published Apr. 25,

1988), hybridization-triggered cleavage agents. (See, e.g., Krol et al. (1988)

BioTechniques 6:958-976) or intercalating agents. (See, e.g, Zon (1988) Pharm. Res. 5:539-549). To this end, the oligonucieotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.

The antisense oligonucieotide may comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouracil, 5- bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxy ethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouricil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1 -methylguanine, 1-methylinosine, 2,2-idimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta- D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio- N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5- methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl- 2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.

The antisense oligonucieotide may also comprise at least one modified sugar moiety selected from the group including but not limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucieotide comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.

In yet a further embodiment, the antisense oligonucieotide is an alpha -anomeric oligonucieotide. An alpha -anomeric oligonucieotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual beta -units, the strands run parallel to each other (Gautier et al. (1987) Nucl. Acids Res. 15:6625-6641). The oligonucieotide is a 2'-0-methylribonucleotide (Inoue et al. (1987) Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

Oligonucleotides of the invention may be synthesized by standard methods known in the art, e.g by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (1988) Nucl. Acids Res. 16:3209), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:7448- 7451), etc.

While antisense nucleotides complementary to the LTM coding region sequence can be used, those complementary to the transcribed untranslated region are most preferred.

The antisense molecules should be delivered to cells which express LTM genes in vivo, and particulary to the hippocampus. A number of methods have been developed for delivering antisense DNA or RNA to cells; e.g., antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systematically.

However, it is often difficult to achieve intracellular concentrations of the antisense sufficient to suppress translation on endogenous mRNAs. Therefore a prefeπed approach utilizes a recombinant DNA construct in which the antisense oligonucieotide is placed under the control of a strong pol III or pol II promoter. The use of such a construct to transfect target cells in the patient will result in the transcription of sufficient amounts of single stranded RNAs that will form complementary base pairs with the endogenous LTM transcripts and thereby prevent translation of the LTM mRNA. For example, a vector can be introduced in vivo such that it is taken up by a cell and directs the transcription of an antisense RNA. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells. Expression of the sequence encoding the antisense RNA can be by any promoter known in the art to act in mammalian, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include but are not limited to: the SV40 early promoter region (Bemoist and Chambon (1981) Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al. (1980) Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al. (1981) Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al. (1982) Nature 296:39-42), etc. Any type of plasmid, cosmid, YAC or viral vector can be used to prepare the recombinant DNA construct which can be introduced directly into the tissue site; e.g., the choroid plexus or hypothalamus. Alternatively, viral vectors can be used which selectively infect the desired tissue; (e.g., for brain, herpesvirus vectors may be used), in which case administration may be accomplished by another route (e.g., systematically).

Ribozyme molecules designed to catalytically cleave LTM mRNA transcripts can also be used to prevent translation of LTM mRNA and expression of LTM (See, e.g., PCT Publication No. WO 90/1 1364, published Oct. 4, 1990; Sarver et al. (1990) Science 247: 1222-1225 and U.S. Pat. No. 5,093,246). While ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy LTM mRNAs, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5'-UG-3'. The construction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach (1988) Nature 334:585-591. There are hundreds of potential hammerhead ribozyme cleavage sites within the nucleotide sequence of human LTM cDNA (FIG. 1). Preferably the ribozyme is engineered so that the cleavage recognition site is located near the 5' end of the LTM mRNA; i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.

The ribozymes of the present invention also include RNA endoribonucleases (hereinafter "Cech-type ribozymes") such as the one which occurs naturally in Tetrahymena thermophila (known as the IVS, or L-19 IVS RNA) and which has been extensively described by Thomas Cech and collaborators (Zaug, et al. (1984) Science 224:574-578; Zaug and Cech (1986) Science 231 :470-475; Zaug, et al. (1986) Nature 324:429-433; published PCT Publication No. WO 88/04300 by University Patents Inc.; Been and Cech, (1986) Cell 47:207-216). The Cech-type ribozymes have an eight base pair active site which hybridizes to a target RNA sequence whereafter cleavage of the target RNA takes place. The invention encompasses those Cech-type ribozymes which target eight base-pair active site sequences that are present in an LTM gene.

As in the antisense approach, the ribozymes can be composed of modified oligonucleotides (e.g. for improved stability, targeting, etc.) and should be delivered to cells which express the LTM gene in vivo. A preferred method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous LTM messages and inhibit translation. Because ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency. Endogenous LTM gene expression can also be reduced by inactivating or "knocking out" the LTM gene or its promoter using targeted homologous recombination, (see, e.g, Smithies et al. (1985) Nature 317:230-234; Thomas and Capecchi (1987) Cell 51:503-512; Thompson et al. (1989) Cell 5:313-321; each of which is incorporated by reference herein in its entirety). For example, a mutant, non-functional LTM (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous LTM gene (either the coding regions or regulatory regions of the LTM gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express LTM in vivo. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the LTM gene. Such approaches are particularly suited in the agricultural field where modifications to ES (embryonic stem) cells can be used to generate animal offspring with an inactive LTM (e g., see Thomas and Capecchi, 1987, and Thompson, 1989, supra). However this approach can be adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors, e.g., herpes virus vectors for delivery to brain tissue; e.g., the hypothalamus and/or choroid plexus.

Alternatively, endogenous LTM gene expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of the LTM gene (i.e., the LTM promoter and/or enhancers) to form triple helical structures that prevent transcription of the LTM gene in target cells in the body. (See generally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84; Helene, C, et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioassays 14(12):807-15).

Likewise, the antisense constructs of the present invention, by antagonizing the normal biological activity of one of the LTM proteins, can be used in the manipulation of issue, e.g. lipid metabolism, both in vivo and for ex vivo tissue cultures.

Furthermore, like the antisense techniques (e.g. microinjection of antisense molecules, or transfection with plasmids whose transcripts are antisense with regard to an LTM mRNA or gene sequence) antagonizing the normal biological activity of one of the LTM proteins can be used to investigate role of LTM in lipid metabolism. Such techniques can be utilized in cell culture, but can also be used in the creation of transgenic animals, as detailed below.

Nucleic acid molecules to be used in triple helix formation for the inhibition of transcription are preferably single stranded and composed of deoxyribonucleotides. The base composition of these oligonucleotides should promote triple helix formation via Hoogsteen base pairing rules, which generally require sizable stretches of either purines or pyrimidines to be present on one strand of a duplex. Nucleotide sequences may be pyrimidine-based, which will result in TAT and CGC triplets across the three associated strands of the resulting triple helix. The pyrimidine-rich molecules provide base complementarity to a purine-rich region of a single strand of the duplex in a parallel orientation to that strand. In addition, nucleic acid molecules may be chosen that are purine-rich, for example, containing a stretch of G residues. These molecules will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority of the purine residues are located on a single strand of the targeted duplex, resulting in CGC triplets across the three strands in the triplex.

Alternatively, the potential sequences that can be targeted for triple helix formation may be increased by creating a so called "switchback" nucleic acid molecule. Switchback molecules are synthesized in an alternating 5'-3', 3'-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizable stretch of either purines or pyrimidines to be present on one strand of a duplex.

Antisense RNA and DNA, ribozyme, and triple helix molecules of the invention may be prepared by any method known in the art for the synthesis of DNA and RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well known in the art such as for example solid phase phosphoramide chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polyinerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.

Moreover, various well-known modifications to nucleic acid molecules may be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone.

E. LTM proteins The present invention also makes available isolated 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 refeπed to herein as "contaminating proteins") or "substantially pure or purified preparations" are defined as encompassing preparations of LTM polypeptides having less than about 20%o (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. By "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%o 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). The term "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. In preferred embodiments, 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 peptidyl portions of LTM proteins can be obtained by screening peptides recombinantly produced from the corresponding fragment of the nucleic acid encoding such peptides. In addition, fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. For example, 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.

Another aspect of the present invention concerns recombinant forms of the LTM proteins. Recombinant polypeptides preferred by the present invention, in addition to native LTM proteins, 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. In a preferred embodiment, 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.

The present invention further pertains to recombinant forms of the subject LTM polypeptides. Such recombinant LTM polypeptides preferably are capable of functioning in one of either role of an agonist or antagonist of at least one biological activity of a wildtype ("authentic") LTM protein.

In general, polypeptides referred to herein as having an activity (e.g., are "bioactive") of an LTM protein mimic or antagonize all or a portion of the biological/biochemical activities of a naturally occurring LTM protein. Examples of such biological activity include the ability to modulate memory consolidation and other fornix-mediated activity in the hippocapmus. According to the present invention, a polypeptide has biological activity if it is a specific agonist or antagonist of a naturally- occurring form of an LTM protein.

The present invention further pertains to methods of producing the subject LTM polypeptides. For example, a host cell transfected with a nucleic acid vector directing expression of a nucleotide sequence encoding the subject polypeptides can be cultured under appropriate conditions to allow expression of the peptide to occur. The cells may be harvested, lysed and the protein isolated. A cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art. The recombinant LTM polypeptide can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for such peptide. In a preferred embodiment, the recombinant LTM polypeptide is a fusion protein containing a domain which facilitates its purification, such as GST fusion protein. Moreover, it will be generally appreciated that, under certain circumstances, it may be advantageous to provide homologs of one of the subject LTM polypeptides which function in a limited capacity as one of either an LTM agonist (mimetic) or an LTM antagonist, in order to promote or inhibit only a subset of the biological activities of the naturally-occurring form of the protein. Thus, specific biological effects can be elicited by treatment with a homolog of limited function, and with fewer side effects relative to treatment with agonists or antagonists which are directed to all of the biological activities of naturally occurring forms of LTM proteins.

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. Alternatively, 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. In addition, agonistic forms of the protein may be generated which are constitutively active. Thus, the LTM protein and homologs thereof provided by the subject invention may be either positive or negative regulators of memory consolidation.

The recombinant LTM polypeptides of the present invention also include homologs of the wild-type LTM proteins, such as versions of those protein which are resistant to proteolytic cleavage, as for example, due to mutations which alter ubiquitination or other enzymatic targeting associated with the protein.

LTM polypeptides may also be chemically modified to create LTM derivatives by forming covalent or aggregate conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like. Covalent derivatives of LTM proteins can be prepared by linking the chemical moieties to functional groups on amino acid sidechains of the protein or at the N-terminus or at the C-terminus of the polypeptide.

Modification of the structure of the subject LTM polypeptides can be for such purposes as enhancing therapeutic or prophylactic efficacy, stability (e.g., ex vivo shelf life and resistance to proteolytic degradation in vivo), or post-translational modifications (e.g., to alter phosphorylation pattern of protein). Such modified peptides, when designed to retain at least one activity of the naturally-occurring lorm of the protein, or to produce specific antagonists thereof, are considered functional equivalents of the LTM polypeptides described in more detail herein. Such modified peptides can be produced, for instance, by amino acid substitution, deletion, or addition.

For example, it is reasonable to expect ihat an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid (i.e. isosteric and/or isoelectric mutations) will not have a major effect on the biological activity of the resulting molecule. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are can be divided into four families: (1) acidic = aspartate, glutamate; (2) basic = lysine, arginine, histidine; (3) nonpolar = alanine, valine, leucine, isoleucine, proline, phenylalamne, methionine, tryptophan; and (4) uncharged polar = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. In similar fashion, the amino acid repertoire can be grouped as (1) acidic = aspartate, glutamate; (2) basic = lysine, arginine histidine, (3) aliphatic = glycine, alanine, valine, leucine, isoleucine, serine, threonine, with serine and threonine optionally be grouped separately as aliphatic-hydroxyl; (4) aromatic = phenylalamne, tyrosine, tryptophan; (5) amide = asparagine, glutamine; and (6) sulfur-containing — cysteine and methionine. (see, for example, Biochemistry, 2nd ed., Ed. by L. Stryer, W. H. Freeman and Co., 1981). Whether a change in the amino acid sequence of a peptide results in a functional LTM homolog (e.g. functional in the sense that the resulting polypeptide mimics or antagonizes the wild-type form) can be readily determined by assessing the ability of the variant peptide to produce a respoise in cells in a fashion similar to the wild-type protein, or competitively inhibit such a response. Polypeptides in which more than one replacement has taken place can readily be tested in the same manner. This invention further contemplates a method for generating sets of combinatorial mutants of the subject LTM proteins as well as truncation mutants, and is especially useful for identifying potential variant sequences (e.g. homologs) that are functional in modulating signal transduction from a lipid receptor. The purpose of screening such combinatorial libraries is to generate, for example, novel LTM homologs which can act as either agonists or antagonist, or alternatively, possess novel activities all together. To illustrate, LTM homologs can be engineered by the present method to provide selective, constitutive activation of a memory consolidation signaling pathway. Thus, combinatorially-derived homologs can be generated to have an increased potency relative to a naturally occurring form of the protein. Likewise, LTM homologs can be generated by the present combinatorial approach to selectively inhibit (antagonize) memory consolidation. For instance, mutagenesis can provide LTM homologs which are able to bind other signal pathway proteins (or DNA) yet prevent propagation of the signal, e.g. the homologs can be dominant negative mutants. Moreover, manipulation of certain domains of LTM by the present method can provide domains more suitable for use in fusion proteins. In one embodiment, the variegated library of LTM variants is generated by combinatorial mutagenesis at the nucleic acid level, and is encoded by a variegated gene library. For example, a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential LTM sequences are expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g. for phage display) containing the set of LTM sequences therein.

There are many ways by which such libraries of potential LTM homologs can be generated from a degenerate oligonucieotide sequence. Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic genes then ligated into an appropriate expression vector. The purpose of a degenerate set of genes is to provide, in one mixture, all of the sequences encoding the desired set of potential LTM sequences. The synthesis of degenerate oligonucleotides is well known in the art (see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp 273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11 :477. Such techniques have been employed in the directed evolution of other proteins (see, for example, Scott et al. (1990) Science 249:386-390; Roberts et al. (1992) Proc. Natl. Acad. Sci. USA 89:2429-2433; Devlin et al. (1990) Science 249: 404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87: 6378-6382; as well as U.S. Pat. Nos. 5,223,409, 5.198,346, and 5,096,815).

A wide range of techniques arc known in the art for screening gene products of combinatorial libraries made by point mutatiors or truncation, and for screening cDNA libraries for gene products having a certain property. Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of LTM homologs. The most widely used techniques for screening large gene libraries typically comprises cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene whose product was detected. Each of the illustrative assays described below are amenable to high through-put analysis as necessary to screen large numbers of degenerate LTM sequences created by combinatorial mutagenesis techniques.

The invention also provides for reduction of the LTM proteins to generate mimetics, e.g. peptide or non-peptide agents, which are able to disrupt binding of an LTM polypeptide of the present invention with either upstream or downstream components of a lipid uptake signaling cascade, such as binding proteins or interactors. Thus, such mutagenic techniques as described above are also useful to map the determinants of the LTM proteins which participate in protein-protein interactions involved in, for example, binding of the subject LTM polypeptide to proteins which may function upstream (including both activators and repressors of its activity) or to proteins or nucleic acids which may function downstream of the LTM polypeptide, whether they are positively or negatively regulated by it, for example. To illustrate, the critical residues of a subject LTM polypeptide which are involved in molecular recognition of, for example, components upstream or downstream of an LTM can be determined and used to generate LTM-derived peptidomimetics which competitively inhibit binding of the authentic LTM protein with that moiety. By employing, for example, scanning mutagenesis to map the amino acid residues of each of the subject LTM proteins which are involved in binding other extracellular proteins, peptidomimetic compounds can be generated which mimic those residues of the LTM protein which facilitate the interaction. Such mimetics may then be used to interfere with the normal function of an LTM protein. For instance, non-hydrolyzable peptide analogs of such residues can be generated using benzodiazepine (e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., see Huffman et al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substituted gamma lactam rings (Garvey et al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), keto-methylene pseudopepi ides (Ewenson et al. (1986) J. Med. Chem. 29:295; and Ewenson et al. in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co. Rockland, 111, 1985), b-turn dipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26:647; and Sato et al. (1986) J. Chem. Soc. Perkin. Trans. 1:1231), and b-aminoalcohols (Gordon et al. (1985) Biochem. Biophys. Res. Commun. 126:419; and Dann et al. (1986) Biochem. Biophys. Res. Commun. 134:71).

F. Cells expression LTM proteins This invention also pertains to host cells transfected to express a recombinant form of the subject LTM polypeptides. The host cell may be any prokaryotic or eukaryotic cell. Thus, 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. Ligating the polynucleotide sequence into a gene construct, such as an expression vector, and transforming or transfecting into hosts, either eukaryotic (yeast, avian, insect or mammalian) or prokaryotic (bacterial cells), are standard procedures used in producing other well-known proteins, e.g. MAP kinase, p53, WT1, PTP phosphatases, SRC, and the like. Similar procedures, or modifications thereof, can be employed to prepare recombinant LTM polypeptides by microbial means or tissue- culture technology in accord with the subject invention.

The recombinant LTM genes can be produced by ligating a nucleic acid encoding an LTM protein, or a portion thereof, into a vector suitable for expression in either prokaryotic cells, eukaryotic cells, or both. Expression vectors for production of recombinant forms of the subject LTM polypeptides include plasmids and other vectors. For instance, suitable vectors for the expression of an LTM polypeptide include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX- derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.

A number of vectors exist for the expression of recombinant proteins in yeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 are cloning and expression vehicles useful in the introduction of genetic constructs into S. cerevisiae (see, for example, Broach el al. (1983) in Experimental Manipulation of Gene Expression, ed. M. Inouye Academic Press, p. 83, incoφorated by reference herein). These vectors can replicate in E. coli due the presence of the pBR322 ori, and in S. cerevisiae due to the replication determinant of the yeast 2 micron plasmid. In addition, drug resistance markers such as ampicillin can be used.

The preferred mammalian expression vectors contain both prokaryotic sequences, to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, PKO-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively. derivatives of viruses such as the bovine papillomavirus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells. The various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989) Chapters 16 and 17.

In some instances, it may be desirable to express the recombinant LTM polypeptide by the use of a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL94 1), pAcUW-derived vectors (such as pAcUWl), and pBlueBac- derived vectors (such as the beta -gal containing pBlueBac III).

When it is desirable to express only a portion of an LTM protein, such as a form lacking a portion of the N-terminus, i.e. a truncation mutant which lacks the signal peptide, it may be necessary to add a start codon (ATG) to the oligonucieotide fragment containing the desired sequence to be expressed. It is well known in the art that a methionine at the N-terminal position can be enzymatically cleaved by the use of the enzyme methionine aminopeptidase (MAP). MAP has been cloned from E. coli (Ben- Bassat et al. (1987) J. Bacteriol. 169:751-757) and Salmonella typhimurium and its in vitro activity has been demonstrated on recombinant proteins (Miller et al. (1987) Proc. Natl. Acad. Sci. 84:2718-1722). Therefore, removal of an N-terminal methionine, if desired, can be achieved either in vivo by expressing LTM-derived polypeptides in a host which produces MAP (e.g., E. coli or CM89 or S. cerevisiae), or in vitro by use of purified MAP (e.g., procedure of Miller et al, supra). In other embodiments transgenic animal, described in more detail below could be used to produce recombinant proteins.

G. Fusion Proteins and Immunogens.

In another embodiment, the coding sequences for the polypeptide can be incoφorated as a part of a fusion gene including a nucleotide sequence encoding a different polypeptide. This type of expression system can be useful under conditions where it is desirable to produce an immunogenic fragment of an LTM protein. For example, the VP6 capsid protein of rotavirus can be used as an immunologic carrier protein for portions of the LTM polypeptide, either in the monomeric form or in the form of a viral particle. The nucleic acid sequences corresponding to the portion of a subject LTM protein to which antibodies are to be raised can be incoφorated into a fusion gene construct which includes coding sequences for a late vaccinia virus structural protein to produce a set of recombinant viruses expressing fusion proteins comprising LTM epitopes as part of the virion. It has been demonstrated with the use of immunogenic fusion proteins utilizing the hepatitis b surface antigen fusion proteins that recombinant hepatitis b virions can be utilized in this role as well. Similarly, chimeric constructs coding for fusion proteins containing a portion of an LTM protein and the poliovirus capsid protein can be created to enhance immunogenicity of the set of polypeptide antigens (see, for example, EP Publication No: 0259149; and Evans et al. (1989) Nature 339:385; Huang et al. (1988) J. Virol. 62:3855; and Schlienger et al. (1992) J. Virol. 66:2).

The Multiple Antigen Peptide system for peptide-based immunization can also be utilized to generate an immunogen, wherein a desired portion of an LTM polypeptide is obtained directly from organo-chemical synthesis of the peptide onto an oligomeric branching lysine core (see, for example, Posnett et al. (1988) J. Biol. Chem. 263:1719 and Nardelli et al. (1992) J. Immunol. 148:914). Antigenic determinants of LTM proteins can also be expressed and presented by bacterial cells.

In addition to utilizing fusion proteins to enhance immunogenicity, it is widely appreciated that fusion proteins can also facilitate the expression of proteins, and accordingly, can be used in the expression of the LTM polypeptides of the present invention. For example, LTM polypeptides can be generated as glutathione-S-transferase (GST-fusion) proteins. Such GST-fusion proteins can enable easy purification of the LTM polypeptide, as for example by the use of glutathione-derivatized matrices (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. (John Wiley & Sons, NY 1991)).

In another embodiment, a fusion gene coding for a purification leader sequence, such as a poly-(His)/enterokinase cleavage site sequence at the N-terminus of the desired portion of the recombinant protein, can allow purification of the expressed fusion protein by affinity chromatography using a Ni2 + metal resin. The purification leader sequence can then be subsequently removed by treatment witn enterokinase to provide the purified protein (e.g., see Hochuli et al. (1987) J. Chromatography 411 :177; and Janknecht et al. PNAS 88:8972).

Techniques for making fusion genes are known to those skilled in the art.

Essentially, the joining of various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt- ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed to generate a chimeric gene sequence (see, for example, Cuπent Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).

H Antibodies

Another aspect of the invention pertains to an antibody specifically reactive with a mammalian LTM protein. For example, by using immunogens derived from an LTM protein, e.g. based on the cDNA sequences, anti-protein/anti-peptide antisera or monoclonal antibodies can be made by standard protocols (See, for example, Antibodies: A Laboratory Manual ed. by Ηarlow and Lane (Cold Spring Harbor Press: 1988)). A mammal, such as a mouse, a hamster or rabbit can be immunized with an immunogenic form of the peptide (e.g., a ammalian LTM polypeptide or an antigenic fragment which is capable of eliciting an antibody response, or a fusion protein as described above). Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art. An immunogenic portion of an LTM protein can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies. In a preferred embodiment, the subject antibodies are immunospecific for antigenic determinants of an LTM protein of a mammal or closely related homologs (e.g. at least 900%> homologous, and more preferably at least 94% homologous).

Following immunization of an animal with an antigenic preparation of an LTM polypeptide, anti-LTM antisera can be obtained and, if desired, polyclonal anti-LTM antibodies isolated from the serum. To produce monoclonal antibodies, antibody- producing cells (lymphocytes) can be harvested from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells. Such techniques are well known in the art, and include, for example, the hybridoma technique (originally developed by Kohler and Milstein, (1975) Nature 256: 495-497), the human B cell hybridoma technique (Kozbar et al. (1983) Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96). Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with a mammalian LTM polypeptide of the present invention and monoclonal antibodies isolated from a culture comprising such hybridoma cells. In one embodiment anti-human LTM antibodies specifically react with any of the proteins encoded by the DNA of ATCC deposit Nos. 98125-98128.

The term "antibody" as used herein is intended to include fragments thereof which are also specifically reactive with one of the subject mammalian LTM polypeptides. Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab)2 fragments can be generated by treating antibody with pepsin. The resulting F(ab)2 fragment can be treated to reduce disulfide bridges to produce Fab fragments. The antibody of the present invention is further intended to inchlde bispecific, single-chain and chimeric molecules having affinity for an LTM protein conferred by at least one CDR region of the antibody. In preferred embodiments, the antibody further comprises a label attached thereto and able to be detected, (e.g. the label can be a radioisotope, fluorescent compound, enzyme or enzyme co-factor). Antibodies which specifically bind LTM epitopes can also be used in immunohistochemical staining of tissue samples in order to evaluate the abundance and pattern of expression of each of the subject LTM polypeptides. Anti-LTM antibodies can be used diagnostically in immuno-precipitation and immuno-blotting to detect and evaluate LTM protein levels in tissue as part of a clinical testing procedure. For instance, such measurements can be useful in predictive valuations of the onset or progression of proliferative disorders. Likewise, the ability to monitor LTM protein levels in an individual can allow determination of the efficacy of a given treatment regimen for an individual afflicted with such a disorder. The level of LTM polypeptides may be measured from cells in bodily fluid, such as in samples of cerebral spinal fluid, such as produced by biopsy. Diagnostic assays using anti-Ti antibodies can include, for example, immunoassays designed to aid in early diagnosis of a degenerative disorder. Diagnostic assays using anti-LTM polypeptide antibodies can also include immunoassays designed to aid in early diagnosis and phenotyping neoplasic or hypeφlastic disorders.

Another application of anti-LTM antibodies of the present invention is in the immunological screening of cDNA libraries constructed in expression vectors such as lambda gtl l, lambda gtl8-23, lambda ZAP, and lambda ORF8. Messenger libraries of this type, having coding sequences inserted in the correct reading frame and orientation, can produce fusion proteins. For instance, lambda gtl l will produce fusion proteins whose amino termini consist of beta -galactosidase amino acid sequences and whose carboxy termini consist of a foreign polypeptide. Antigenic epitopes of an LTM protein, e.g. other orthologues of a particular LTM protein or other paralogues from the same species, can then be detected with antibodies, as, for example, reacting nitrocellulose filters lifted from infected plates with anti-LTM antibodies. Positive phage detected by this assay can then be isolated from the infected plate. Thus, the presence of LTM homologs can be detected and cloned from other animals, as can alternate isoforms (including splicing variants) from humans.

I. Drug Screening Assays

Another aspect of the present invention relates to the use of LTM genes and LTM gene products for carrying out assays designed to identify agents which, by modulating the function of certain LTM genes, can be used to modify long term memory consolidation in animals. As described in further detail below, 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. As described in the examples, the LTM genes can range from cell surface receptors and secreted proteins to transcription factors. Accordingly, 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. A variety of assay formats will suffice and, in light of the present inventions, will be comprehended by a skilled artisan.

Monitoring the influence of compounds on cells may be applied not only in basic drug screening, but also in clinical trials. In such clinical trials, the expression of a panel of genes may be used as a "read out" of a particular drug's therapeutic effect.

(i) Cell-Free Assays

In many drug screening programs which test libraries of compounds and natural extracts, high throughput assays are desirable in order to maximize the number of compounds surveyed in a given period of time. Assays which are performed in cell-free systems, such as may be derived with purified or semi-purified proteins, are often preferred as "primary" screens in that they can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test compound. Moreover, the effects of cellular toxicity and/or bioavailability of the test compound can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the drug on the molecular target as may be manifest in an alteration of binding affinity with upstream or downstream elements or with intrinsic enzymatic activity. Many of the LTM proteins identified by the subject method will be amenable to some form of cell-free assay formats. Soluble proteins, 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.

Accordingly, in an exemplary screening assay of the present invention, 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". Examples of 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. Moreover, 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.

Complex formation between the 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. Examples include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix. For example, 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. Following incubation, 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. Alternatively, 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.

Other techniques for immobilizing proteins or nucleic acids on matrices are also available for use in the subject assay. For instance, either LTM or its cognate binding partner can be immobilized utilizing conjugation of biotin and streptavidin. For instance, 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). Alternatively, antibodies reactive with the LTM protein, but which do not interfere with binding of upstream or downstream binding partners, can be derivatized to the wells of the plate, and the LTM protein trapped in the wells by antibody conjugation. As above, 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, in addition to those described above for the GST-immobilized 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. In the instance of the latter, the enzyme can be chemically conjugated or provided as a fusion protein with an LTM- IP. To illustrate, 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. 3,3'-diamino-benzadine tetrahydrochloride or 4-chloro-l-napthol. Likewise, 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). For processes which rely on immunodetection for quantitating one of the proteins trapped in the complex, antibodies against the protein, such as anti-LTM antibodies, can be used. Alternatively, 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). For instance, 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. 266:21150-21157) which includes a 10-residue sequence from c-myc, as well as the pFLAG system (International Biotechnologies, Inc.) or the pEZZ-protein A system (Pharmacia, N.J.). Other 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. For instance, 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.

In preferred embodiments, 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.

(ii) Cell Based Assays In addition to cell-free assays, such as described above, 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. For instance, where the LTM protein is a receptor, the assay can be used to identify compounds which bind to the receptor or effect the ability of the receptor to bind its ligand. In other embodiments, cells which are phenotypically sensitive to the presence or activity of the LTM protein, e.g., if it produces a moφhological 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. As with the cell-free assays, agents which produce a statistically significant change in LTM-dependent responses (either inhibition or potentiation) can be identified. For example, the level of expression of genes or gene products which are up- or downregulated in response to the presence or activity of an LTM protein can be detected. In preferred embodiments, 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.

In the event that the LTM proteins themselves, or in complexes with other proteins, are capable of binding DNA and modifying transcription of a gene, a transcriptional based assay could be used, for example, in which an LTM-responsive regulatory sequence is operably linked to a detectable marker gene.

In yet another aspect of the invention, 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. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and PCT Publication No. WO 94/10300). Briefly, the two hybrid assay relies on reconstituting in vivo a functional transcriptional activator protein from two separate fusion proteins. In particular, the method makes use of chimeric genes which express hybrid proteins. To illustrate, a first chimeric 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.

a. Host Cells

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. Examples of 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.

If yeast cells are used, the yeast 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. The term "yeast", as used herein, includes not only yeast in a strictly taxonomic sense, i.e., unicellular organisms, but also yeast-like multicellular fungi or filamentous fungi.

The choice of appropriate host cell will also be influenced by the choice of detection signal. For instance, reporter constructs, as described below, 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. In other embodiments, second messenger generation can be measured directly in the detection step, such as mobilization of intracellular calcium or phospholipid metabolism are quantitated. In yet other embodiments indicator genes can be used to detect receptor-mediated signaling.

b. Exemplary LTM proteins: Enzymes In certain embodiments, the LTM protein will include an intrinsic enzymatic activity which, when potentiated or inhibited, can alter LTM performance in the animal. For example, the LTM protein may include a protease activity (e.g., serine protein, cysteine protease, aspartic acid protease, metalloprotease), a kinase activity, a lipase activity, a phosphatase activity, permease activity, a ligase activity, a ubquitin ligating activity, a glycoslation or deglycosylation activity, or the like. In general, there exists natural and synthetic substrates for most ezymatic activities. Accordingly, the subject invention specifically contemplates the an assay utilizing the LTM, or an enzymatically active fragment thereof, and a substrate of the activity in order to identify compounds which potentiate or inhibit the activity.

c. Exemplary LTM proteins: Cytokine Receptors

In certain embodiments, the LTM gene identified in the assays described above will encode a receptor that is up- or donw-regulated as part of fornix-mediated memory consolidation. Most of the cytokine receptors that constitute distinct superfamilies do not possess intrinsic protein tyrosine kinase domains, yet receptor stimulation usually invokes rapid tyrosine phosphorylation of intracellular proteins, including the receptors themselves. Many members of the cytokine receptor superfamily acitvate 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. For example, cytokine receptors can cause the phosphorylation of signal transducers and activators of transcription (STAT) proteins STATlα, STAT2β, and STAT3, as well as of two STAT-related proteins, p94 and p95. The STAT proteins translocate to the nucleus and bind to specific DNA sequences, thus suggesting a mechanism by which cytokines may activate speicfic 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.

d. Exemplary LTM proteins: Nuclear Receptors

Another class of receptors which may be up- or down-regulated in the hippocampus are the nuclear receptors. The nuclear receptors may be viewed as ligand- dependent transcription factors. These receptors provide a direct link between extracellular signals, mainly hormones, and transcriptional responses. Their transcriptional activation fuction is regulated by endogenous small molecules, such as steroid hormones, vitamin D, ecdysone, retinoic acids and thyroid hormones, which pass readily through the plasma membrane and bind their receptors inside the cell (Laudet and Adelmant (1995) Current Biology 5:124). The majority of these receptors appear to contain three domains: a variable amino terminal domain; a highly conserved, DNA- binding domain and a moderately conserved, carboxyl-terminal ligand-binding domain (Power et al. (1993) Curr. Opin. Cell Biol. 5:499-504). In certain embodiments, the subject assay may be derived to utilize a hormone- dependent reporter construct for selection. For instance, transcriptional response elements which bind a nuclear receptor can be used to drive expression of reporter construct in response to ligand binding to the receptor. Such response elements are enhancer-like DNA sequences that confer ligand responsiveness via interaction with the nuclear receptor. See, for example, U.S. Patents 5,298,429 and 5,071,773, both to Evans, et. al. Moreover, the art describes the functional expression of such receptors in yeast. See also for example, Caplan et al. (1995) J Biol Chem 270:5251-7; and Baniahmad et al. (1995) Mol Endocrinol 9: 34-43.

e. Exemplary LTM proteins: Receptor Tyrosine kinases

In still another embodiment, the LTM gene may encode a receptor tyrosine kinase. The receptor tyrosine kinases can be divided into five subgroups on the basis of structural similarities in their extracellular domains and the organization of the tyrosine kinase catalytic region in their cytoplasmic domains. Sub-groups I (epidermal growth factor (EGF) receptor-like), II (insulin receptor-like) and the eph/eck family contain cysteine-rich sequences (Hirai et al., (1987) Science 238:1717-1720 and Lindberg and Hunter, (1990) Mol. Cell. Biol. 10:6316-6324). The functional domains of the kinase region of these three classes of receptor tyrosine kinases are encoded as a contiguous sequence ( Hanks et al. (1988) Science 241:42-52). Subgroups III (platelet-derived growth factor (PDGF) receptor-like) and IV (the fibro-blast growth factor (FGF) receptors) are characterized as having immunoglobulin (Ig)-like folds in their extracellular domains, as well as having their kinase domains divided in two parts by a variable stretch of unrelated amino acids (Yanden and Ullrich (1988) supra and Hanks et ΆX. (X9%&) supra). In certain embodiments, the LTM protein may be a receptor of the EPH family. The expression patterns determined for some of the EPH family receptors have implied important roles for these molecules in development and maintance of adult CNS tissue. As used herein, the terms "EPH receptor" or "EPH-type receptor" refer to a class of receptor tyrosine kinases, comprising at least eleven paralogous genes, though many more orthologs exist within this class, e.g. homologs from different species. EPH receptors, in general, are a discrete group of receptors related by homology and easily reconizable, e.g., they are typically characterized by an extracellular domain containing a characteristic spacing of cysteine residues near the N-terminus and two fibronectin type III repeats (Hirai et al. (1987) Science 238:1717-1720; Lindberg et al. (1990) Mol Cell Biol 10:6316-6324; Chan et al. (1991) Oncogene 6:1057-1061; Maisonpierre et al. (1993) Oncogene 8:3277-3288; Andres et al. (1994) Oncogene 9:1461-1467; Henkemeyer et al. (1994) Oncogene 9:1001-1014; Ruiz et al. (1994) Mech Dev 46:87- 100; Xu et al. (1994) Development 120:287-299; Zhou et al. (1994) J Neurosci Res 37:129-143; and references in Tuzi and Gullick (1994) Br J Cancer 69:417-421). Exemplary EPH receptors include the eph, elk, eck, sek, mek4, hek, hek2, eek, erk, tyrol, tyro4, tyro5, tyro6, tyrol 1, cek4, cek5, cekό, cek7, cek8, cek9, ceklO, bsk, rtkl, rtk2, rtk3, mykl, myk2, ehkl, ehk2, pagliaccio, htk, erk and nuk receptors. The term "EPH receptor" refers to the membrane form of the receptor protein, as well as soluble extracellular fragments which retain the ability to bind the ligand of the present invention.

In exemplary embodiments, 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.

f. Exemplary LTM proteins: G Protein-Coupled Receptors.

One family of signal transduction cascades found throughout the CNS utilizes heterotrimeric "G proteins." Many different G proteins are known to interact with receptors. G protein signaling systems include three components: the G protein coupled receptor ("GCR"), a GTP-binding protein (G protein), and an intracellular target protein. Thus, in certain embodiments of the subject drug screening assay, it is anticipated that the LTM is a GCR or a G protein.

In their resting state, the G proteins, which consist of alpha (α), beta (β) and gamma (γ) subunits, are complexed with the nucleotide guanosine diphosphate (GDP) and are in contact with receptors. When a homione or other first messenger binds to receptor, the receptor changes conformation and this alters its interaction with the G protein. This spurs the α subunit to release GDP, and the more abundant nucleotide guanosine triphosphate (GTP), replaces it, activating the G protein. The G protein then dissociates to separate the α subunit from the still complexed beta and gamma subunits. Either the Gα subunit, or the Gβγ complex, depending on the pathway, interacts with an effector. The effector (which is often an enzyme) in turn converts an inactive precursor molecule into an active "second messenger," which may diffuse through the cytoplasm, triggering a metabolic cascade. After a few seconds, the Gα converts the GTP to GDP, thereby inactivating itself. The inactivated Gα may then reassociate with the Gβγ complex.

Hundreds, if not thousands, of receptors convey messages through heterotrimeric G proteins, of which at least 17 distinct forms have been isolated. Although the greatest variability has been seen in the α subunit, several different β and γ structures have been reported. There are, additionally, several different G protein-dependent effectors. Most G protein-coupled receptors are comprised of a single protein chain that is threaded through the plasma membrane seven times. Such receptors are often referred to as seven-transmembrane receptors (STRs). More than a hundred different STRs have been found, including many distinct receptors that bind the same ligand, and there are likely many more STRs awaiting discovery. Exemplary G-protein coupled receptors which may be identified as LTM genes include, but are not limited to, dopaminergic, muscarinic cholinergic, α-adrenergic, β-adrenergic, opioid (including delta and mu), cannabinoid, serotoninergic, and GABAergic receptors. The LTM protein of the present assays may be a G protein-coupled receptor, and may be recombinantly expressed in a cell which is to be genetically engineered for the puφose of the present assays.

Known ligands for G protein coupled receptors include: purines and nucleotides, such as adenosine, cAMP, ATP, UTP, ADP, melatonin and the like; biogenic amines (and related natural ligands), such as 5-hydroxytryptamine, acetylcholine, doparnine, adrenaline, adrenaline, adrenaline., histamine, noradrenaline, noradrenaline, noradrenaline., tyramine/octopamine and other related compounds; peptides such as adrenocorticotrophic hormone (acth), melanocyte stimulating hormone (msh), melanocortiiis, neurotensin (nt), bombesin and related peptides, endothelins, cholecystokinin, gastrin, neurokinin b (nk3), invertebrate tachykinin-like peptides, substance k (nk2), substance p (nkl), neuropeptide y (npy), thyrotropin releasing-factor (trf), bradykinin, angiotensin ii, beta-endoφhin, c5a anaphalatoxin, calcitonin, chemokines (also called intercrines), corticotrophic releasing factor (erf), dynoφhin, endoφhin, fmlp and other formylated peptides, follitropin (fsh), fungal mating pheremones, galanin, gastric inhibitory polypeptide receptor (gip), glucagon-like peptides (glps), glucagon, gonadotropin releasing hormone (gnrh), growth hormone releasing hormone(ghrh), insect diuretic hormone, interleukin- 8, leutropin (lh/hcg), met- enkephalin, opioid peptides, oxytocin, parathyroid hormone (pth) and pthφ, pituitary adenylyl cyclase activiating peptide (pacap), secretin, somatostatin, thrombin, thyrotropin (tsh), vasoactive intestinal peptide (vip), vasopressin, vasotocin; eicosanoids such as ip-prostacyclin, pg-prostaglandins, tx-thromboxanes; retinal based compounds such as vertebrate 11-cis retinal, invertebrate 11-cis retinal and other related compounds; lipids and lipid-based compounds such as cannabinoids, anandamide, lysophosphatidic acid, platelet activating factor, leukotrienes and the like; excitatory amino acids and ions such as calcium ions and glutamate. Thus, the test compound may be, in certain preferred embodiments, a compound which is an anlaog of such ligands.

g. Exemplary Screening and Selection Assays: 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 receptor- or ion channel-regulated, including adenylyl cyclase, cyclic GMP, phosphodiesterases, phosphoinositidase C, and phospholipase A , as well as a variety of ions.

In one embodiment, the GTPase enzymatic activity by G proteins can be measured in plasma membrane preparations by determining the breakdown of γ32p QTP using techniques that are known in the art (For example, see Signal Transduction: A Practical Approach. G. Milligan, Ed. Oxford University Press, Oxford England). When receptors that modulate cAMP are tested, it will be possible to use standard techniques for cAMP detection, such as competitive assays which quantitate [^H]cAMP in the presence of unlabelled cAMP.

Certain receptors and ion channels 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. The other product of PIP2 breakdown, 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 known techniques, including, for example, the generation of arachadonate in the cell. In the case of certain receptors and ion channels, it may be desirable to screen for changes in cellular phosphorylation. Such assay formats may be useful when the receptor of interest is a receptor kinase or phosphatase. For example, immunoblotting (Lyons and Nelson (1984) Proc. Natl. Acad. Sci. USA 81 :7426-7430) using anti- phosphotyrosine, anti-phosphoserine or abti-phosphothreonine antibodies. In addition, tests for phosphorylation could be also useful when the receptor itself may not be a kinase, but activates protein kinases or phosphatase that function downstream in the signal transduction pathway.

One such cascade is the 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 ρ44 and p42 MAP kinases (ERK1 and ERK2). Activated MAP kinase 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).

In yet another embodiment, the signal transduction pathway of the targeted receptor or ion channel 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 altenatively, by using a substrate which produces a detectable product. In prefeπed embodiments, 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. In an illustrative embodiment 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. (1992) Anal Biochem 204:96-102. For example, 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. Upon cleavage of the peptide, however, the quencher is separated from the donor group, leaving behind a fluorescent fragment. Thus, activation of the enzyme results in cleavage of the detection peptide, and dequenching of the fluorescent group.

In still other embodiments, 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. For example , 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). As an exemplary method of 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.

As certain embodiments described above suggest, in addition to directly measuring second messenger production, 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 a part of a completely heterologous construct, e.g., as part of a reporter gene construct.

In one embodiment, the indicator gene is an unmodified endogenous gene. In certain instances, it may be desirable to increase the level of transcriptional activation of the endogenous indicator gene by the signal pathway in order to, for example, improve the signal-to-noise of the test system, or to adjust the level of response to a level suitable for a particular detection technique. In one embodiment, 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. In other embodiments, 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. In general, manipulation of the genomic sequence for the indicator gene can be carried out using techniques known in the art, including homologous recombination.

In another exemplary embodiment, 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. Again, using such techniques as homologous recombination, the regulatory sequence can be so altered at the genomic locus of the indicator gene.

In still another embodiment, 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. h. Exemplary Screening and Selection Assays: Reporter Genes

Examples of reporter genes include, but are not limited to CAT (chloramphenicol acetyl transferase) (Alton and Vapnek (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. 182: 231-238, Hall et al. (1983) J. Mol. Appl. Gen. 2: 101), human placental secreted alkaline phosphatase (Cullen and Malim (1992) Methods in Enzymol. 216:362-368).

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 prefeπed 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.

Other promoters and transcriptional control elements, in addition to those described above, include the vasoactive intestinal peptide (VIP) gene promoter (cAMP responsive; Fink et al. (1988), Proc. Natl. Acad. Sci. 85:6662-6666); the somatostatin gene promoter (cAMP responsive; Montminy et al. (1986), Proc. Natl. Acad. Sci. 8.3:6682-6686); the proenkephalin promoter (responsive to cAMP, nicotinic agonists, and phorbol esters; Comb et al. (1986), Nature 323:353-356); the phosphoenolpyruvate carboxy-kinase gene promoter (cAMP responsive; Short et al. (1986), J. Biol. Chem. 261:9721-9726); the NGFI-A gene promoter (responsive to NGF, cAMP, and serum; Changelian et al. (1989). Proc. Natl. Acad. Sci. 86:377-381); and others that may be known to or prepared by those of skill in the art. In the case of receptors which modulate cyclic AMP, 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). When this serine residue is phosphorylated, CREB binds to a recognition sequence known as a CRE (cAMP Responsive Element) found to the 5' of promotors known to be responsive to elevated cAMP levels. Upon binding of phosphorylated CREB to a CRE, transcription from this promoter is increased.

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.

Therefore, 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.

As described in Bonni et al. (1993) Science 262:1575-1579, the observation that CNTF treatment of SK-N-MC cells leads to the enhanced interaction of STAT/p91 and STAT related proteins with specific DNA sequences suggested that these proteins might be key regulators of changes in gene expression that are triggered by CNTF. Consistent with this possibility is the finding that DNA sequence elements similar to the consensus DNA sequence required for STAT/p91 binding are present upstream of a number of genes previously found to be induced by CNTF (e.g., Human c-fos, Mouse c-fos, Mouse tisl l, Rat junB, Rat SOD-1, and CNTF). Those authors demonstrated the ability of STAT/p91 binding sites to confer CNTF responsiveness to a non-responsive reporter gene. Accordingly, a reporter construct for use in the present invention for detecting signal transduction through STAT proteins, such as from cytokine receptors, 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. In preferred embodiments, 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. The term "receptor-responsive promoter" indicates a promoter which is regulated by some product of the target receptor's signal transduction pathway.

Alternatively, the promoter may be one which is repressed by the receptor pathway, thereby preventing expression of a product which is deleterious to the cell. With 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 incoφorating a suitable operator site into the promoter or other suitable region of the marker gene.

J. Pharmaceutical Preparations of Identified Agents After identifying certain test compounds in the subject assay ,e.g., as potentiators or inhibitors of memory consolidation, the practioner of the subject assay will continue to test the efficacy and specificity of the selected compounds both in vitro and in vivo. Whether for subsequent in vivo testing, or for administration to an animal as an approved drug, 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. The optimum concentration of the active ingredient(s) in the chosen medium can be determined empirically, according to procedures well known to medicinal chemists. As used herein, "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. Except insofar as any conventional media or agent is incompatible with the activity of the compound, its use in the pharmaceutical preparation of the invention is contemplated. 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. In the case of freeze-dried preparations, supporting 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).

In certain embodiment, the pharmaceutical of the present invention is a gene delivery system for gene therapy with a therapeutic LTM gene. Such gene therapy systems can be introduced into a patient by any of a number of methods, each of which is familiar in the art. For instance, 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. In other embodiments, initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized. For example, 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. Alternatively, where the complete gene delivery system can be produced intact from recombinant cells, e.g. retroviral vectors, the pharmaceutical preparation can comprise one or more cells which produce the gene delivery system.

K. Methods of Treatment

In various embodiments, 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. 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. In other embodiments, 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.

In certain embodiments, 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 known as retrograde amnesia. The inability to form new memories is known as anterograde amnesia.

Complaints of memory problems are common. Poor concentration, poor arousal and poor attention all may disrupt the memory process to a degree. The subjective complaint of memory problems therefore must be distinguished from true amnesias. This is usually done at the bedside in a more gross evaluation and through specific neuropsychological test. Defects in visual and verbal memory can be separated through such test. In amnesias there is by definition a preservation of other mental capacities such as logic. The neurobiologic theory of memory described above would predict that amnesias would have relatively few pathobiologic variations. Clinically the problem of amnesias often presents as a result of a sudden illness in an otherwise healthy person.

Exemplary forms of 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 Heφes simplex encephalitis .

(i) Effective Dose

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%o 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. For any compound used in the method of the invention, 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. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

L. Diagnostic and Prognostic Assays

The present method also provides a method for determining if a subject is at risk for a disorder characterized deterioration of memory consolidation . In prefeπed embodiments, 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. To illustrate, 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, (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) abeπant 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. As set out below, the present invention provides a large number of assay techniques for detecting lesions in an LTM gene, and importantly, provides the ability to discern between different molecular causes underlying a disorder.

In an exemplary embodiment, there is provided a nucleic acid composition comprising a (purified) oligonucieotide probe including a region of nucleotide sequence which is capable of hybridizing to a sense or antisense sequence of an LTM gene, or naturally occuπing 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.

In preferred embodiments, 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. To illustrate, 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. As set out below, the present invention provides a large number of assay techniques for detecting lesions in LTM genes.

In certain embodiments, 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. (1994) PNAS 91 :360-364), the latter of which can be particularly useful for detecting point mutations in the LTM gene (see Abravaya et al. (1995) Nuc Acid Res 23:675-682). In a merely illustrative embodiment, 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. It is anticipated that PCR and or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.

Another embodiment of the invention provides for a nucleic acid composition comprising a (purified) oligonucieotide probe including a region of nucleotide sequence which is capable of hybridizing to a sense or antisense sequence of an LTM gene, or naturally occuπing 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 oligonucieotide 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 known to one of skill in the art, including but not limited to 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 incoφorated herein by reference in its entirety.

This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody (see below) coupled with light microscopic, flow cytometric, or fluorimetric detection. 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. Through the use of such a procedure, it is possible to determine not only the presence of the LTM protein, but also its distribution in the examined tissue. Using the present invention, one of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.

Often a solid phase support or carrier is used as a support capable of binding an antigen or an antibody. Well-known 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 puφoses 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. Thus, 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. Alternatively, 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. One means for labeling an anti-LTM protein specific antibody is via linkage to an enzyme and use in an enzyme immunoassay (EIA) (Voller, "The Enzyme Linked Immunosorbent Assay (ELISA)", Diagnostic Horizons 2:1-7, 1978, Microbiological Associates Quarterly Publication, Walkersville, Md.; Voller, et al., J. Clin. Pathol. 31 :507-520 (1978); Butler, Meth. Enzymol. 73:482-523 (1981); Maggio, (ed.) Enzyme Immunoassay, CRC Press, Boca Raton, Fla., 1980; Ishikawa, et al., (eds.) Enzyme Immunoassay, Kgaku Shoin, Tokyo, 1981). 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 chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.

Detection may also be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect frngeφrint gene wild type or mutant peptides through the use of a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incoφorated by reference herein). The radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.

It is also possible to label the antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emitting metals such as ¹⁵²Eu, 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). 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. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. Likewise, 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 puφoses of labeling are luciferin, luciferase and aequorin.

Moreover, it will be understood that any of the above methods for detecting alterations in an LTM gene or gene product can be used to monitor the course of treatment or therapy.

M. Transgenic Animals

These systems may be used in a variety of applications. For example, the cell- and animal-based model systems may be used to further characterize LTM genes and proteins. In addition, such assays may be utilized as part of screening strategies designed to identify compounds which are capable of ameliorating disease symptoms. Thus, the animal- and cell-based models may be used to identify drugs, pharmaceuticals, therapies and interventions which may be effective in treating disease.

One aspect of the present invention concerns transgenic animals which are 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. In preferred embodiments, 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. In the present invention, 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. Toward this and, tissue-specific regulatory sequences and conditional regulatory sequences can be used to control expression of the transgene in certain spatial patterns. Moreover, temporal patterns of expression can be provided by, for example, conditional recombination systems or prokaryotic transcriptional regulatory sequences.

Genetic techniques which allow for the expression of transgenes can be regulated via site-specific genetic manipulation in vivo are known to those skilled in the art. For instance, genetic systems are available which allow for the regulated expression of a recombinase that catalyzes the genetic recombination a target sequence. As used herein, the phrase "target sequence" refers to a nucleotide sequence that is genetically recombined by a recombinase. The target sequence is flanked by recombinase recognition sequences and is generally either excised or inverted in cells expressing recombinase activity. Recombinase catalyzed recombination events can be designed such that recombination of the target sequence results in either the activation or repression of expression of one of the subject LTM proteins. For example, excision of a target sequence which interferes with the expression of a recombinant LTM gene, such as one which encodes an antagonistic homolog or an antisense transcript, can be designed to activate expression of that gene. This interference with expression of the protein can result from a variety of mechanisms, such as spatial separation of the LTM gene from the promoter element or an internal stop codon. Moreover, the transgene can be made wherein the coding sequence of the gene is flanked by recombinase recognition sequences and is initially transfected into cells in a 3' to 5' orientation with respect to the promoter element. In such an instance, inversion of the target sequence will reorient the subject gene by placing the 5' end of the coding sequence in an orientation with respect to the promoter element which allow for promoter driven transcriptional activation.

The transgenic animals of the present invention all include within a plurality of their cells a transgene of the present invention, which transgene alters the phenotype of the "host cell". Since it is possible to produce transgenic organisms of the invention utilizing one or more of the transgene constructs described herein, a general description will be given of the production of transgenic organisms by referring generally to exogenous genetic material. This general description can be adapted by those skilled in the art in order to incoφorate specific transgene sequences into organisms utilizing the methods and materials described below.

In an illustrative embodiment, either the cre/loxP recombinase system of bacteriophage PI (Lakso et al. (1992) Proc. Natl. Acad. Sci. USA 89:6232-6236; Orban et al. (1992) Proc. Natl. Acad. Sci. USA 89:6861-6865) or the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251 :1351-1355; PCT Publication No. WO 92/15694) can be used to generate in vivo site-specific genetic recombination systems. Cre recombinase catalyzes the site-specific recombination of an intervening target sequence located between loxP sequences. loxP sequences are 34 base pair nucleotide repeat sequences to which the Cre recombinase binds and are required for Cre recombinase mediated genetic recombination. The orientation of loxP sequences determines whether the intervening target sequence is excised or inverted when Cre recombinase is present (Abremski et al. (1984) J. Biol. Chem. 259:1509-1514); catalyzing the excision of the target sequence when the loxP sequences are oriented as direct repeats and catalyzes inversion of the target sequence when loxP sequences are oriented as inverted repeats. Accordingly, genetic recombination of the target sequence is dependent on expression of the Cre recombinase. Expression of the recombinase can be regulated by promoter elements which are subject to regulatory control, e.g., tissue-specific, developmental stage-specific, inducible or repressible by externally added agents. This regulated control will result in genetic recombination of the target sequence only in cells where recombinase expression is mediated by the promoter element. Thus, the activation expression of a recombinant LTM protein can be regulated via control of recombinase expression.

Use of the cre/loxP recombinase system to regulate expression of a recombinant

LTM protein requires the construction of a transgenic animal containing transgenes encoding both the Cre recombinase and the subject protein. Animals containing both the Cre recombinase and a recombinant LTM gene can be provided through the construction of "double" transgenic animals. A convenient method for providing such animals is to mate two transgenic animals each containing a transgene, e.g., an LTM gene and recombinase gene. One advantage derived from initially constructing transgenic animals containing an LTM transgene in a recombinase-mediated expressible format derives from the likelihood that the subject protein, whether agonistic or antagonistic, can be deleterious upon expression in the transgenic animal. In such an instance, a founder population, in which the subject transgene is silent in all tissues, can be propagated and maintained. Individuals of this founder population can be crossed with animals expressing the recombinase in, for example, one or more tissues and/or a desired temporal pattern. Thus, the creation of a founder population in which, for example, an antagonistic LTM transgene is silent will allow the study of progeny from that founder in which disruption of LTM mediated induction in a particular tissue or at certain developmental stages would result in, for example, a lethal phenotype.

Similar conditional transgenes can be provided using prokaryotic promoter sequences which require prokaryotic proteins to be simultaneous expressed in order to facilitate expression of the LTM transgene. Exemplary promoters and the corresponding trans-activating prokaryotic proteins are given in U.S. Pat. No. 4,833,080. Moreover, expression of the conditional transgenes can be induced by gene therapy-like methods wherein a gene encoding the trans-activating protein, e.g. a recombinase or a prokaryotic protein, is delivered to the tissue and caused to be expressed, such as in a cell-type specific manner. By this method, an LTM transgene could remain silent into adulthood until "turned on" by the introduction of the trans- activator.

In an exemplary embodiment, 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. In addition, 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-2^b , H-2^d or H-21 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) .

In one embodiment, the transgene construct is introduced into a single stage embryo. The zygote is the best target for micro-injection. In the mouse, the male pronucleus reaches the size of approximately 20 micrometers in diameter which allows reproducible injection of l-2pl of DNA solution. The use of zygotes as a target for gene transfer has a major advantage in that in most cases the injected DNA will be incoφorated into the host gene before the first cleavage (Brinster et al. (1985) Proc. Natl. Acad. Sci. USA 82:4438-4442). As a consequence, all cells of the transgenic animal will carry the incoφorated transgene. This will in general also be reflected in the efficient transmission of the transgene to offspring of the founder since 50%> of the germ cells will harbor the transgene. ormally, fertilized embryos are incubated in suitable media until the pronuclei appear. At about this time, the nucleotide sequence comprising the transgene is introduced into the female or male pronucleus as described below. In some species such as mice, the male pronucleus is prefeπed. It is most preferred that the exogenous genetic material be added to the male DNA complement of the zygote prior to its being processed by the ovum nucleus or the zygote female pronucleus. It is thought that the ovum nucleus or female pronucleus release molecules which affect the male DNA complement, perhaps by replacing the protamines of the male DNA with histones, thereby facilitating the combination of the female and male DNA complements to form the diploid zygote.

Thus, it is preferred that the exogenous genetic material be added to the male complement of DNA or any other complement of DNA prior to its being affected by the female pronucleus. For example, the exogencus genetic material is added to the early male pronucleus, as soon as possible after the formation of the male pronucleus, which is when the male and female pronuclei are well separated and both are located close to the cell membrane. Alternatively, the exogenous genetic material could be added to the nucleus of the sperm after it has been induced to undergo decondensation. Sperm containing the exogenous genetic material can then be added to the ovum or the decondensed sperm could be added to the ovum with the transgene constructs being added as soon as possible thereafter.

Introduction of the transgene nucleotide sequence into the embryo may be accomplished by any means known in the art such as, for example, microinjection, electroporation, or lipofection. Following introduction of the transgene nucleotide sequence into the embryo, the embryo may be incubated in vitro for varying amounts of time, or reimpianted into the suπogate host, or both. In vitro incubation to maturity is within the scope of this invention. One common method in to incubate the embryos in vitro for about 1-7 days, depending on the species, and then reimplant them into the surrogate host.

For the puφoses of this invention a zygote is essentially the formation of a diploid cell which is capable of developing into a complete organism. Generally, the zygote will be comprised of an egg containing a nucleus formed, either naturally or artificially, by the fusion of two haploid nuclei from a gamete, or gametes. Thus, the gamete nuclei must be ones which are naturally compatible, i.e., ones which result in a viable zygote capable of undergoing differentiation and developing into a functioning organism. Generally, a euploid zygote is preferced. If an aneuploid zygote is obtained, then the number of chromosomes should not vary by more that one with respect to the euploid number of the organism from which either gamete originated.

In addition to similar biological considerations, physical ones also govern the amount (e.g., volume) of exogenous genetic material which can be added to the nucleus of the zygote or to the genetic material which forms a part of the zygote nucleus. If no genetic material is removed, then the amount of exogenous genetic material which can be added is limited by the amount which will be absorbed without being physically disruptive. Generally, the volume of exogenous genetic material inserted will not exceed about 10 picoliters. The physical effects of addition must not be so great as to physically destroy the viability of the zygote. The biological limit of the number and variety of DNA sequences will vary depending upon the particular zygote and functions of the exogenous genetic material and will be readily apparent to one skilled in the art, because the genetic material, including the exogenous genetic material, of the resulting zygote must be biologically capable of initiating and maintaining the differentiation and development of the zygote into a functional organism.

The number of copies of the transgene constructs which are added to the zygote is dependent upon the total amount of exogencus genetic material added and will be the amount which enables the genetic transformation to occur. Theoretically only one copy is required; however, generally, numerous copies are utilized, for example, 1,000-20,000 copies of the transgene construct, in order to insure that one copy is functional. As regards the present invention, there will often be an advantage to having more than one functioning copy of each of the inserted exogenous DNA sequences to enhance the phenotypic expression of the exogenous DNA sequences. Any technique which allows for the addition of the exogenous genetic material into nucleic genetic material can be utilized so long as it is not destructive to the cell, nuclear membrane or other existing cellular or genetic structures. The exogenous genetic material is preferentially inserted into the nucleic genetic material by microinjection. Microinjection of cells and cellular structures is known and is used in the art.

Reimplantation is accomplished using standard methods. Usually, the surrogate host is anesthetized, and the embryos are inserted into the oviduct. The number of embryos implanted into a particular host will vary by species, but will usually be comparable to the number of off spring the species naturally produces. Transgenic offspring of the surrogate host may be screened for the presence and/or expression of the transgene by any suitable method. Screening is often accomplished by Southern blot or Northern blot analysis, using a probe that is complementary to at least a portion of the transgene. Western blot analysis using an antibody against the protein encoded by the transgene may be employed as an alternative or additional method for screening for the presence of the transgene product. Typically, DNA is prepared from tail tissue and analyzed by Southern analysis or PCR for the transgene. Alternatively, the tissues or cells believed to express the transgene at the highest levels are tested for the presence and expression of the transgene using Southern analysis or PCR, although any tissues or cell types may be used for this analysis. Alternative or additional methods for evaluating the presence of the transgene include, without limitation, suitable biochemical assays such as enzyme and/or immunological assays, histological stains for particular marker or enzyme activities, flow cytometric analysis, and the like. Analysis of the blood may also be useful to detect the presence of the transgene product in the blood, as well as to evaluate the effect of the transgene on the levels of various types of blood cells and other blood constituents.

Progeny of the transgenic animals may be obtained by mating the transgenic animal with a suitable partner, or by in vitro fertilization of eggs and/or sperm obtained from the transgenic animal. Where mating with a partner is to be performed, the partner may or may not be transgenic and/or a knockout; where it is transgenic, it may contain the same or a different transgene, or both. Alternatively, the partner may be a parental line. Where in vitro fertilization is used, the fertilized embryo may be implanted into a suπogate host or incubated in vitro, or both. Using either method, the progeny may be evaluated for the presence of the transgene using methods described above, or other appropriate methods. The transgenic animals produced in accordance with the present invention will include exogenous genetic material. As set out above, the exogenous genetic material will, in certain embodiments, be a DNA sequence which results in the production of an LTM protein (either agonistic or antagonistic), and antisense transcript, or an LTM mutant. Further, in such embodiments the sequence will be attached to a transcriptional control element, e.g., a promoter, which preferably allows the expression of the transgene product in a specific type of cell.

Retroviral infection can also be used to introduce transgene into a non-human animal. The developing non-human embryo can be cultured in vitro to the blastocyst stage. During this time, the blastomeres can be targets for retroviral infection (Jaenich, R. (1976) Proc. Natl. Acad. Sci USA 73:1260-1264). Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Manipulating the Mouse Embryo, Hogan eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1986). The viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner et al. (1 985) Proc. Natl. Acad. Sci. 82:6927-6931 ; Van der Putten et al. (1985) Proc. Natl. Acad. Sci. USA 82:6148-6152). Transfection is easily and efficiently obtained by culturing the blastomeres on a monolayer of virus-producing cells (Van der Putten, supra; Stewart et al. (1987) EMBO J. 6:383-388). Alternatively, infection can be performed at a later stage. Virus or virus-producing cells can be injected into the blastocoele (Jahner et al. (1982) Nature 298: 623-628). Most of the founders will be mosaic for the transgene since incoφoration occurs; only in a subset of the cells which formed the transgenic non-human animal. Further, the founder may contain various retroviral insertions of the transgene at different positions in the genome which generally will segregate in the offspring. In addition, it is also possible to introduce transgenes into the germ line by intrauterine retroviral infectior of the midgestation embryo (Jahner et al., supra).

A third type of target cell for transgene introduction is the embryonal stem cell (ES). ES cells are obtained from pre-implani:ation embryos cultured in vitro and fused with embryos (Evans et al. (1981) Nature 292:154-156; Bradley et al. (1984) Nature 309:255-258; Gossler et al. (1986) Proc. Natl. Acad. Sci. USA 83:9065-9069; and Robertson et al. (1986) Nature 322:445-448). Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retrovirus-mediated transduction. Such transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal. For review see Jaenisch, R. (1988) Science 240:1468- 1474. In one embodiment, 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. 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 aπanged 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.

Generally, the embryonic stem cells (ES cells) used to produce the knockout animals will be of the same species as the knockout animal to be generated. Thus for example, mouse embryonic stem cells will usually be used for generation of knockout mice.

Embryonic stem cells are generated and maintained using methods well known to the skilled artisan such as those described by Doetschman et al. (1985) J. Embryol. Exp. Moφhol. 87:27-45). Any line of ES cells can be used, however, the line chosen is typically selected for the ability of the cells to integrate into and become part of the germ line of a developing embryo so as to create germ line transmission of the knockout construct. Thus, any ES cell line that is believed to have this capability is suitable for use herein. One mouse strain that is typically used for production of ES cells, is the 129J strain. Another ES cell line is murine cell line D3 (American Type Culture Collection, catalog no. CKL 1934) Still another prefened ES cell line is the WW6 cell line (loffe et al. (1995) Proc. Natl. Acad. Sci. USA 92:7357-7361). The cells are cultured and prepared for knockout construct insertion using methods well known to the skilled artisan, such as those set forth by Robertson in: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. IRL Press, Washington, D.C., 1987); by Bradley et al. (1986) Cmrent Topics in Devel. Biol. 20:357-371); and by Hogan et al. (Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

Insertion of the knockout construct into the ES cells can be accomplished using a variety of methods well known in the art including for example, electroporation, microinjection, and calcium phosphate treatment. A prefened method of insertion is electroporation.

Each knockout construct to be inserted into the cell must first be in the linear form. Therefore, if the knockout construct has beer inserted into a vector (described infra), linearization is accomplished by digesting the DNA with a suitable restriction endonuclease selected to cut only within the vector sequence and not within the knockout construct sequence.

For insertion, the knockout construct is added to the ES cells under appropriate conditions for the insertion method chosen, as is known to the skilled artisan. Where more than one construct is to be introduced into the ES cell, each knockout construct can be introduced simultaneously or one at a time.

If the ES cells are to be electroporated, the ES cells and knockout construct DNA are exposed to an electric pulse using an electroporation machine and following the manufacturer's guidelines for use. After electroporation, the ES cells are typically allowed to recover under suitable incubation conditions. The cells are then screened for the presence of the knockout construct.

Screening can be accomplished using a variety of methods. Where the marker gene is an antibiotic resistance gene, for example, the ES cells may be cultured in the presence of an otherwise lethal concentration of antibiotic. Those ES cells that survive have presumably integrated the knockout construct. If the marker gene is other than an antibiotic resistance gene, a Southern blot of the ES cell genom DNA can be probed with a sequence of DNA designed to hybridize only to the marker sequence. Alternatively, PCR can be used. Finally, if the marker gene is a gene that encodes an enzyme whose activity can be detected (e.g., b-galactosidase), the enzyme substrate can be added to the cells under suitable conditions, and the enzymatic activity can be analyzed. One skilled in the art will be familiar with other useful markers and the means for detecting their presence in a given cell. All such markers are contemplated as being included within the scope of the teaching of this invention.

The knockout construct may integrate into several locations in the ES cell genome, and may integrate into a different location in each ES cell's genome due to the occunence of random insertion events. The desired location of insertion is in a complementary position to the DNA sequence to be knocked out, e.g., the LTM coding sequence, transcriptional regulatory sequence, etc. Typically, less than about l-5%> of the ES cells that take up the knockout construct will actually integrate the knockout construct in the desired location. To identify those ES cells with proper integration of the knockout construct, total DNA can be extracted from the ES cells using standard methods. The DNA can then be probed on a Southern blot with a probe or probes designed to hybridize in a specific pattern to genomic DNA digested with particular restriction enzyme(s). Alternatively, or additionally, the genomic DNA can be amplified by PCR with probes specifically designed to amplify DNA fragments of a particular size and sequence (i.e., only those cells containing the knockout construct in the proper position will generate DNA fragments of the proper size).

After suitable ES cells containing the knockout construct in the proper location have been identified, the cells can be inserted into an embryo. Insertion may be accomplished in a variety of ways known to the skilled artisan, however a prefened method is by microinjection. For microinjection, about 10-30 cells are collected into a micropipette and injected into embryos that are at the proper stage of development to permit integration of the foreign ES cell containing the knockout construct into the developing embryo. For instance, as the appended Examples describe, the transformed ES cells can be micro injected into blastocysts. The suitable stage of development for the embryo used for insertion of ES cells is very species dependent, however for mice it is about 3.5 days. The embryos are obtained by perfusing the uterus of pregnant females. Suitable methods for accomplishing this are known to the skilled artisan, and are set forth by, e.g., Bradley et al. (supra).

While any embryo of the right stage of development is suitable for use, prefened embryos are male. In mice, the prefened embryos also have genes coding for a coat color that is different from the coat color encoded by the ES cell genes. In this way, the offspring can be screened easily for the presence of the knockout construct by looking for mosaic coat color (indicating that the ES cell was incoφorated into the developing embryo). Thus, for example, if the ES cell line carries the genes for white fur, the embryo selected will carry genes for black or brown fur.

After the ES cell has been introduced into the embryo, the embryo may be implanted into the uterus of a pseudopregnant foster mother for gestation. While any foster mother may be used, the foster mother is typically selected for her ability to breed and reproduce well, and for her ability to care for the young. Such foster mothers are typically prepared by mating with vasectomized males of the same species. The stage of the pseudopregnant foster mother is important for successful implantation, and it is species dependent. For mice, this stage is about 2-3 days pseudopregnant.

Offspring that are born to the foster mother may be screened initially for mosaic coat color where the coat color selection strategy (as described above, and in the appended examples) has been employed. In addition, or as an alternative, DNA from tail tissue of the offspring may be screened for the presence of the knockout construct using Southern blots and/or PCR as described above. Offspring that appear to be mosaics may then be crossed to each other, if they are believed to carry the knockout construct in their germ line, in order to generate homozygous knockout animals. Homozygotes may be identified by Southern blotting of equivalent amounts of genomic DNA from mice that are the product of this cross, as well as mice that are known heterozygotes and wild type mice.

Other means of identifying and characterizing the knockout offspring are available. For example, Northern blots can be used to probe the mRNA for the presence or absence of transcripts encoding either the gene knocked out, the marker gene, or both. In addition, Western blots can be used to assess the level of expression of the LTM gene knocked out in various tissues of the offspring by probing the Western blot with an antibody against the particular LTM protein, or an antibody against the marker gene product, where this gene is expressed. Finally, in situ analysis (such as fixing the cells and labeling with antibody) and/or FACS (fluorescence activated cell sorting) analysis of various cells from the offspring can be conducted using suitable antibodies to look for the presence or absence of the knockout construct gene product.

Yet other methods of making knock-out or disruption transgenic animals are also generally known. See, for example, Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Recombinase dependent knockouts can also be generated, e.g. by homologous recombination to insert target sequences, such that tissue specific and/or temporal control of inactivation of an LTM gene can be controlled by recombinase sequences (described infra).

Animals containing more than one knockout construct and/or more than one transgene expression construct are prepared in any of several ways. The prefened manner of preparation is to generate a series of mammals, each containing one of the desired transgenic phenotypes. Such animals are bred together through a series of crosses, backcrosses and selections, to ultimately generate a single animal containing all desired knockout constructs and/or expression constructs, where the animal is otherwise congenic (genetically identical) to the wild type except for the presence of the knockout construct(s) and/or transgene(s) .

The present invention is further illustrated by the following examples which should not be construed as limiting in any way. The contents of all cited references (including literature references, issued patents, published patent applications as cited throughout this application are hereby expressly incoφorated by reference.

Exemplification

(i) Methods Surgery. Sixty- four male, Long Evans rats weighing between 200 and 250 grams were used in the behavioral experiments. Animals were housed iu individual wire mesh cages and maintained in a 12 hr on/12 hr off light dark cycle. All rats were allowed free access to food and water. Rats were anesthetized with sodium pentobarbital (55 mg/kg, I. P.), and placed in a stereotaxic apparatus, where a midline incision was made and the scalp retracted to expose the skull. Electrolytic lesions of the fornix were made by drilling holes through the skull at 0.4 and 1.4 mm posterior to Bregma and 0.6 and 1.0 mm lateral to the midline. Monopolar electrodes (Teflon-coated wire, 125 m in diameter) were lowered at each site to a depth of 4.4 mm. measured from the surface of the skull. DC cuπent at 1 mA was passed through the electrodes for a duration of 12 s. The electrodes were then removed, and the wound sutured. Control animals received sham operations in which holes were drilled in the skull overlying the fornix. The electrode was then inserted and withdrawn without passing cunent. Postoperatively, the animals received a prophylactic dose of antibiotic (Claforan, 0.1 ml, I.M.), and were kept warm and monitored until spontaneous movement occuned. Once stabilized, they were returned to their home cages and left to recover for 7 days prior to behavioral testing.

Inhibitory Avoidance Training. The inhibitory avoidance chamber consisted of a rectangular shaped perspex box, divided into two compartments, the safe and the shock compartments. The safe compartment (measuring 21 (L) x 24.5 (H) x 17 (W) cm) was white and illuminated by a light fixture, fastened to the cage lid. The shock compartment (measuring 30.5 (L) x 20.3 (H) x 21.5 (W) cm) was dark and made of black perspex. Foot shocks were delivered to the grid floor of this chamber via a constant current scrambler circuit. The two compartments were separated by an automatically operated sliding door. The apparatus was located in a sound attenuated, non-illuminated room. During training sessions, each rat was placed in the safe compartment with its head facing away from the door. After a period of ten seconds, the door was automatically opened, allowing the rat access to the shock chamber. The door closed one second after the rat entered the shock chamber, and a brief footshock (1.5 mA for 2 s) was administered to the rat. Latency to enter the shock chamber was taken as a measure of acquisition. The rat was then removed from the apparatus and returned to its home cage. Retention tests were performed either immediately (0 hours), or at 6, 24 or 48 hours later by placing the rat back in the safe compartment and measuring the latency to enter the shock chamber. Footshock was not administered on the retention test, and testing was terminated at 540 seconds. Statistical analysis of the behavioral data was performed using two way ANOVA followed by Student Newman-Keuls post-hoc tests.

Following completion of behavioral testing, rats were deeply anesthetized with sodium pentobarbital and transcardially perfused with 0.9% saline followed by 10% formalin. The brains were dissected and stored in a sucrose-formalin solution until being sliced into 40 m sections on a freezing microtome. The slices were mounted on glass slides and stained with Cresyl violet and evaluated for accuracy of the fornix lesion.

For all biochemical analyses, rats received a single training trial in the inhibitory avoidance apparatus as described above. Following training, rats were either sacrificed immediately (0 hours), or returned to their home cages and then sacrificed 3, 6, or 9 hours later. In each of the biochemical experiments there were four groups of animals: normal unoperated rats which received training on the inhibitory avoidance task as described above (shock); unoperated rats which were exposed to the inhibitory avoidance apparatus but which did not receive a footshock (no shock); rats with lesions of the fornix, which were trained on the task (fornix-shock) and rats with lesions of the fornix which were exposed to the apparatus but did not receive a footshock (fornix-no shock). At each timepoint, brains from each of these four conditions were rapidly dissected and frozen for Western blot analysis or perfused for immunohistochemistry as described below. Western Blot Analysis. Anti-PCPEB (Ser-133) and anti-CREB polyclonal antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). Extracts from rat hippocampi were obtained by polytron homogenizafion in cold lysis buffer with protease inhibitors (0.2 M NaCl, 0.1 M Hepes, 10% glycerol, 2 mM NaF, 2 mM Na₄P₂O₇, 5 mM EDTA, ImM EGTA, 2 mMDTT, 0.5 mM PMSF, 1 mM benzamidine, 10 mg/ml leupeptin, 400 U/ml aprotinin, 1 mM microcystin). After 10 min on ice, the samples were centrifuged at 16,000 g for 15 min at 4 C. The supematants were collected and their total protein concentration determined using the BioRad Protein Assay (BioRad Laboratories, Hercules, CA). The lysates were then ahquoted and stored at -80 C. Equal amounts of total protein conesponding to 25 g/lane were resolved on denaturing 10% SDS-PAGE gels and transfened to Immobilon-P (PVDF) transfer membranes (Millipore, Bedford, MA) by electroblotting. Membranes were pretreated with 5% BLOTTO buffer and then incubated with anti-PCRBB (1/2000) or anti-CREB (1/1000) antisera. in Tris-buffered saline (TBS) overnight at 4 C. The membranes were then washed with TBS, treated with a secondary HRP-labeled donkey anti-rabbit antibody (1/4000) in TBS for 1 hr, washed again and incubated with HRP-streptavidin complex and ECL detection reagents (Amersham, Arlington Heights, Illinois). Membranes were exposed to BioMax MS film (Eastman Kodak, Rochester, NY) and quantitative densitometric analysis was performed using NIH Image. Statistical analysis was performed using one-way and two-way ANOVA followed by Student Newman- Keuls or Dunnett Two-Tailed post-hoc analysis. Immunohistochemistry. Animals were perfused transcardially with cold phosphate-buffered saline (PBS) containing 20 U/ml heparin (Sigma, St. Louis, MO), followed by cold 4% paraformaldehyde in PBS. Brains were post-fixed overnight in the same fixative with 30% sucrose, and then cryoprotected overnight in 30% sucrose/PBS. Forty m sections were cut in the coronal plane on a freezing microtome. Immunostaining was performed on free-floating slices using, the streptavidin-biotin complex immunoperoxidase technique according to manufacturer's instructions (ImmunoPure ABC Peroxidase Rabbit IgG Staining Kit, Pierce, Rockford, IL). Briefly, sections underwent a series of pre-incubations in 0.3% hydrogen peroxide, 0.3%o Triton X-100, and 10%> normal goat serum). The slices were incubated with P-CREB antibody diluted at 1/1000 for 48 h at 4 C, washed 3 times with PBS and then treated with a 1/400 dilution of biotinylated goat anti-rabbit IgG in PBS for 30 min at room temperature. Slices were then washed 3 times in PBS and incubated with avidin- biotinylated ERP. Staining was revealed by incubating the slices in 0.25 mg/ml diaminebenzidene (Sigma) at room temperature for 5-8 min. After washing with water the slices were mounted on gelatin-coated slides, air-dried and lightly counterstained with Cresyl violet.

(ii) Results

Lesions of the fornix disrupt consolidation of a long-term memory. The fornix is a massive fiber bundle connecting the hippocampus with the septum and hypothalamus. Previous work has shown an impairment in long-term memory following inhibitory avoidance training in animals with fornix lesions.¹⁴ We set out to determine in more detail the nature of this deficit, Rats received electrolytic lesions of the fornix and, following recovery from surgery, were tested for memory acquisition and retention in the inhibitory avoidance task.

Figure la shows a photomicrograph of a representative fornix lesion. In 25 out of the 32 lesion rats, the dorsal fornix was severed and the fimbria was extensively damaged. In addition to damage to the fornix and fimbria, the anterior aspects of the lateral and triangular septa] nuclei and the septofimbrial nucleus were at least partially damaged in all subjects. The remaining 7 animals had partial damage to the fornix and fimbria, and only minor damage to the septal nuclei. No damage to the underlying thalamic structures or to the hippocampal formation was observed in any case.

In the inhibitory avoidance task, animals are asked to form an association of a location with an aversive stimulus. They demonstrate memory by avoiding the negatively reinforced location. In our experiments, training consisted of placing a rat in the lighted chamber of a box with connected lighted and dark chambers. A door was opened between the chambers, allowing the rat to follow its natural tendency to avoid light and cross into the dark chamber. Once in the dark side of the box, the animals received a foot shock (1.5 mA for 2 sec). Memory retention was measured as the tendency for the animals to avoid the dark side in subsequent trials.

Figure lb illustrates the effects of fornix lesions on acquisition and retention of the single-trial inhibitory avoidance task, with retention delays of 0, 6, 24 and 48 hours. As can be seen from the figure, control rats and rats with lesions of the fornix did not differ in terms of acquisition of the task. Latencies to enter the shock chamber for the first time were similar for the two groups of animals, and did not differ statistically (p > 0.05).

The two groups of animals did, however, differ in their overall retention profiles. At short delay intervals (0 and 6 hours after training), control animals and rats with lesions of the fornix showed very similar levels of retention. Analysis of variance indicated that at these short delay intervals, the two groups did not differ from one another (p > 0.05). In contrast, there was a clear difference between the, performance of the two groups at longer (24 and 48 hours) retention intervals. Analysis of variance revealed a significant difference between the control and fornix groups at 24 and 48 hours (F(l, 28) = 18.268, p < 0-0002). This difference appears to reflect an increase in retention latencies for control animals with no apparent change in retention latencies for rats with fornix lesions. Thus, although lesions of the fornix do not result in a total amnesia for the task (as indicated by the increase in latencies from the acquisition trial to the retention trials), it does appear that the fornix lesions disrupt a time-dependent consolidation process whereby memories become stronger and more resilient over time.

Long-term memory formation is associated with a persistent increase in CREB phosphorylation in hippocampal neurons

To determine where in the brain CREB activation occurs in response to inhibitory avoidance learning, we performed quantitative western blot analysis of phosphorylated CREB (PCREB) in proteins extracted from hippocampus, amy-dala, cortex and cerebellum. PCREB was measured 0, 15, 30 and 60 min following training and in untrained controls. Hippocampi of trained animals showed the highest and most consistent induction of PCREB at all the time points as compared to controls. Increases were more modest and variable in amygdala and cortex, while no, changes were detected in cerebellum (data not shown). Therefore, we focused our investigation on the changes in CREB phosphorylation in the hippocampus.

Groups of control (unoperated) rats received a single training trial on the inhibitory avoidance task and the levels of PCREB were measured by western blot analysis in the hippocampus 0, 3, 6 and 9 hr after training. As shown in Fig. 2a (top) and 2b (open symbols), hippocampal PCREB was increased by 152.3 ± 12.4% in unoperated shock animals immediately after training as compared to the control rats that entered the dark chamber but received no shock. This increase in PCREB was persistent at three (158.6 ± 8.3%), six (155.1 ± 12.4%) and nine (150.7 ± 228.6%) hours after training. A one way ANOVA revealed a significant main effect of time (F(4,26) = 3.346, p < 0.02), and Dunnet post hoc comparisons confirmed that PCREB in these animals was significantly greater at 0, 3 and 6 hours after training as compared to the control no shock group (p < 0.05). Western blot immunostaining of the same samples with an antiserum that detects CREIB protein demonstrated that there was no significant change after training (Fig. 2a, top; 2c, open symbols). Thus, we conclude that the training specifically caused phosphorylation of hippocampal CREB protein.

To determine whether the increase in PCREB was related to the consolidation of the inhibitory avoidance task Or to other stimuli evoked by the environment of the training apparatus, we analyzed PCREB levels in hippocampi of unoperated animals that had been exposed to the inhibitory avoidance chamber but never received the shock (Fig. 2d). No significant changes in CREB phosphorylation at 3 (80.3 ± 6.31%), 6 (101.6 ± 8.9%), or 9 hours (107.5 ± 12.2%) after exposure to the apparatus were found as compared to the no shock group sacrificed immediately following exposure to the testing apparatus (p > 0.05). This suggests that the persistent increase in PCREB was specific for the consolidation of inhibitory avoidance memory.

Immunohistochemistry was performed to determine which cells in the hippocampus respond io training with an increase in PCREB. Immunostaining in the hippocampus of untrained animals revealed low levels of PCREB (e.g. Fig. 3a). Variable staining was observed in the neurons of dentate gyrus and CA3, while CA1 neurons were generally PCREB negative. Inhibitory avoidance training, however, led to a strong and regionally specific increase in PCREB immunostaining. Most striking, were the increases in stained neurons in CA1 and dentate gyrus (e.g. Fig. 3b), although modest increases could also be detected in the CA3 region (data not shown). The increases in PCREB immunoreactivity could be observed immediately after training (Fig. 3a, b) and they persisted in these same populations of neurons at 3 and 6 hrs after training (data not shown).

Fornix lesions block the training-induced increase in hippocampal CREB phosphorylation. We next investigated if the normal PCREB response occurs following inhibitory avoidance training in animals that have fornix lesions. Hippocampi of untrained rats with fornix lesions showed basal levels of PCREB (117.4 ± 4.6% of control) comparable to those found in unoperated no shock controls. However, in striking contrast to unoperated control animals, the levels of CREB phosphorylation in hippocampi of rats with fornix lesions did not significantly increase at 0 (108.7 ± 11.2%) or at 6 hours (80.1 ± 6.7%) after training (Fig. 2a, bottom; 2b, filled symbols). A two-way analysis of variance confirmed a significant difference between the unoperated trained and fornix trained groups (F(5,28) = 6.713 = 0-0003). Student Newman Keuls post hoc analysis revealed that the fornix shock group showed significantly less CREB phosphorylation at 0 and 6 hr after training as compared to the unoperated shock group (p < 0.05). The total amount of CREB protein in the hippocampi of fomix lesion animals was not different from controls, and like controls it did not change up to 6 hrs following training (Fig. 2c, filled symbols). Thus, fomix lesions produce a selective deficit in CREB phosphorylation by inhibitory avoidance training. To determine whether fomix lesions blocked selectively CREB phosphorylation in a specific subset of neurons of the hippocampus, we carried out immunohistochemical studies on serial brain sections of fornix lesion animals before and immediately after training. Representative sections from these experiments, using and PCREB antiserum are shown in Fig. 3c, d. Confirming our data obtained by western blot analysis, no induction of PCREB was found in any hippocampal subregions in rats with fomix lesions. Thus, normal hippocampal CREB activation by inhibitory avoidance training fails to occur in animals with fomix lesions.

(iii) Discussion

Our experiments show that inhibitory avoidance training leads to a spatially restricted increase in the phosphorylation of the transcription factor CREB in the brain. Both the CREB response in the hippocampus and the consolidation of long-term memory depend upon the integrity of the fomix. Thus, the data suggest that some lesions of the temporal lobe memory system produce amnesia by interfering with CREB regulated gene expression required for long-term storage of information.

Hippocampal CREB phosphorylation and inhibitory avoidance memory

In species ranging from fruit flies to mice, it has been shown that consolidation o/^" long-term synaptic plasticity and memory requires gene expression that is regulated by CREB." Activation of gene expression by CREB requires phosphorylation at Ser- 133. Thus, detection of the sites of CREB phosphorylation in the brain may reveal the neural circuits involved in storage of specific types of memory. We find that neurons in the CAI region and dentate gyrus selectively respond to inhibitory avoidance training with an increase in CREB phosphorylation. These PCREB data are in excellent conespondence with those obtained very recently by Impey, et al.¹⁵ showing an increase in CRE-mediated gene expression in hippocampal neurons following inhibitory avoidance training in transgenic mice. Thus, taken together, the data strongly suggest That a subset of hippocampal neurons participate in the storage of inhibitory avoidance memory.

The increase in hippocampal PCREB was long-lasting (>6 hrs). This finding suggests that persistent activation of CREB-mediated gene expression is important for consolidation of the memory trace. Consistent with this idea, memory can be disrupted by intrahippocampal injection of protein kinase A inhibitors during a period lasting from 3-6 hrs after training. Conversely, memory is enhanced by injections of PKA activators during the same time period.^{16 17}

Inhibitory avoidance memory occurs with a single trial, and it is remarkably long-lasting. The memory is created by pairing a specific location with an aversive stimulus (a foot shock). We found that the simple exploration of the new environment was not sufficient to trigger the PCREB response in hippocampus; it required the concunent shock. However, we did not investigate the effect of shock alone. We reasoned that shock would trigger associations with whatever sensory stimuli are present in the environment at the time of the shock (i.e. a "flashbulb" memory). Thus, we felt there was no reason to assume a priori that shock would not produce the PCREB response in hippocampus even if the animal was in an environment other than the dark chamber of the training apparatus. Nonetheless, it is interesting to note that Impey et al.¹⁵ did find that if animals are well-habituated to the training apparatus, then shock alone does not stimulate the increase in CRE-mediated gene expression in hippocampus.

Fornix control of CREB phosphorylation and memory consolidation

The effect of fomix damage on the consolidation of long-term inhibitory avoidance memory closely approximates the consequences of temporal lobe damage on various forms of declarative memory³. We found that although initial learning and performance were unimpaired in fo ix lesion animals, a severe memory deficit was apparent 24 hrs after learning. Thus, this simple paradigm may be a useful model to address the larger question of bow the temporal lobe memory system functions to regulate information storage.

The hippocampal PCREB response to inhibitory avoidance training was deficient in animals with fomix lesions. Given the evidence cited above that CREB phosphorylation and activation, of gene expression is required for consolidation of long- term memory, our data suggest that the effects of fomix lesions on memory could be accounted for entirely by the absence of the normal PCREB response. However, it is interesting to note that despite the complete lack of CREB phosphorylation in the hippocampus, fomix lesion animals were able to leam and retain information for many hours. Even at 24 hrs post-training, fomix lesion animals displayed some memory of the inhibitory avoidance experience, despite their amnesia. The initial learning and memory could reflect synaptic modifications that occur even in the absence of new protein synthesis¹⁸. The residual memory at 24 hrs could be accounted for by changes in structures other than the hippocampus, or by the regulation of gene expression by mechanisms other than CREB phosphorylation. In any case, the fomix lesions help to define more clearly the consequence on memory of a selective deficit in the hippocampal PCREB response.

Why do fomix lesions disrupt the PCREB response and the consolidation of long-term inhibitory avoidance memory? The fomix contains axons that connect the hippocampus with the septum, hypothalamus and brainstem. A reasonable working hypothesis is that the signals that regulate CREB-dependent gene expression in hippocampus ascend via the fomix. Indeed, if activity in the fomix is temporarily arrested immediately prior to inhibitory avoidance training, the consequence on memory is as severe as a complete fomix lesion¹⁹. However, inactivation of the fomix prior to testing memory retention 48 hrs after training has no effect. Thus, activity in the fomix is necessary for consolidation, but not expression, of the memory. This result contrasts with the effects of inactivating the dorsal hippocampus, which prevents both memory encoding and retrieval²⁰.

Clinical significance

Initial learning is likely to result from changes in the transmission of synapses conveying information about where the animal is in space. Whether or not these changes are made permanent depends on the timely occuπence of new gene expression. We propose that signals impinging on hippocampal neurons via the fomix contribute to memory consolidation by modulating the gene expression required for the establishment of long-term memory. Identification of the critical chemical signals and their transduction pathways may suggest treatments for amnesia associated with damage to the temporal lobe memory system.

REFERENCES

14. de Castro, J. & Hall, T.W. Fomix lesions: Effects on active and passive avoidance behavior. Physiol. Psychol. 3, 201-204 (1975).

15. Impey, S., Smith, D. M., Obrietan, K., Donahue, R., Wade,. C. & Storm, D. R. Stimulation of cAMP response element (CRE)-mediated transcription during contextual learning. Nature Neurosci 1, 595-601 (1998).

16. Bemabeu, R., Bevilaqua, L., Ardenghi, P., Bromberg, E, Schmitz, P., Bianchin, M., Izquierdo, I. & Medina, J.H. Involvement of hippocampal cAMP/cAMP-dependent protein kinase signaling pathways in a late memory consolidation phase of aversively motivated learning in rats. Proc. Nad. Acad. Sci. USA 94, 7041-7046 (1997).

17. Izquierdo, I. & Medina, J.H. Memory formation: The sequence of biochemical events in the hippocampus and its connection to activity in other brain areas. Neurobiol. Learn. Mem. 68, 285-316 (1997).

18. Frey, U. & Morris, R.G.M. Synaptic tagging: synapse specificity during protein synthesis-dependent long-term potentiation. Nature. (1997).

19. Baldi, E., Lorenzini, C.N., Sacchetti, B., Tassoni, G. & Bucherelli, C. Entorhinal cortex and fimbria-fomix role in rat's passive avoidance response memorization. Brain Res. 799, 270-277 (1998).

20. Lorenzini, C.A., Baldi, E., Bucherelli, C, Sacchetti, B. & Tassoni, G. Role of dorsal hippocampus in acquisition, consolidation and retrieval of rat's passive avoidance response: a tetrodotoxin functional inactivation study. Brain Res. 730, 32-39 (1996).

Claims

We Claim:

1. A method of identifying an agent which enhances memory consolidation in a mammal, comprising (i) providing a non-human mammal which has been manipulated to create at least partial disruption of fomix-mediated afferant signalling to the hippocampus, said disruption affecting memory consolidation,

(ii) conditioning the mammal with a learning or memory regimen which results in learned behavior in the mammal in the absence of the fomix lesion;

(iii) administering, to the mammal, a test agent to be assessed for effects on memory consolidation; and

(iv) ascertaining the effect of the test agent on the the learned behavior of the mammal; wherein an increase in learned behavior, relative to the absence of the test agent, indicates that the test agent enhances memory consolidation.

2. The method of claim 1, wherein the fomix lesion is generated by mechanical disruption of at least a portion of the fomix.

3. The method of claim 1, wherein the fomix lesion is generated by chemical disruption of at least a portion of the fomix.

4. The method of claim 1, 2 or 3, wherein the fornix lesion is generated by selective dismption of one or more neuronal types.

5. The method of claim 4, wherein the fomix lesion is generated by selective dismption of one or more the neurons selected from the group consisting of fomix cholinergic neurons, fomix GABAergic neurons and fomix serotonergic neurons.

6. The method of claim 1 , wherein the mammal is a rodent.

7. The method of claim 1, wherein the mammal is a transgenic animal.

8. The method of claim 1, wherein the test agent is a organic molecule having a molecular weight less than 2500 amu..

9. The method of claim 1 , which is repeated for a plurality of different test agents.

10. The method of claim 9, which is repeated for at least 10 different test agents.

11. A pharmaceutical preparation comprising, a compound identied in the assay of claim 1, formulated in a pharmaceutically acceptable excipient.

12. A method of identifying an agent which promotes transmission of the afferant signal to the hippocampus in a mammal having undergone dismption of the afferant signal to the hippocampus comprising: (i) subjecting the mammal to a learning and memory test to provide a learned behavior in the mammal;

(ii) administering to the mammal the agent to be assessed; and

(iii) determining the ability of the mammal to retain the learned behavior after completion of a learning phase; wherein if the mammal retains the learned behavior after at least about 24 hours, the agent promotes transmission of the afferant signal to the hippocampus.

13. The method of claim 12, wherein transmission of the afferant signal comprises fo ix-mediated signal transmission.

14. A method of identifying an agent which promotes memory consolidation in a mammal comprising the steps of:

(i) administering to a mammal an agent to be assessed, wherein the mammal has been subjected to a learning and memory test to provide a learned behavior in the mammal;

(ii) determining the extent of phosphorylation of CREB in the hippocampus of the mammal; and

(iii) comparing the extent of phosphorylation of CREB in the hippocampus of the mammal to the extent of phosphorylation of CREB in an untrained control mammal, wherein if the extent of phosphorylation of CREB in the mammal is greater than the extent of phosphorylation of CREB in the control mammal, the agent promotes memory consolidation.

15. The method of claim 14, wherein the mammal has been manipulated to create at least partial dismption of fomix-mediated afferant signalling to the hippocampus, said dismption affecting memory consolidation

16. A method of identifying an agent to treat amnesia caused by dismption of memory consolidation, comprising:

(i) subjecting a mammal having undergone dismption of the afferant signal to the hippocampus to a learning and memory test to provide a learned behavior in the mammal;

(ii) administering to the mammal the agent to be assessed; and

(iii) determining the ability of the mammal to retain the learned behavior after completion of a learning phase, wherein if the mammal retains the learned behavior after at least about 24 hours, the agent can be used to treat amnesia cuased by dismption of memory consolidation.

17. A method for the identification of mammalian genes involved in memory consolidation comprising comparing the level of expression of genes from a control animal, characterized by having undergone memory consolidation or being untrained, with the level of expression of genes from a test animal, characterized by having at least partial dismption of fomix-mediated afferant signalling to the hippocampus, said dismption affecting memory consolidation having dismption of the afferant signal to the hippocampus, wherein genes that are up-or downregulated in the test animal relative to the the control animal are candidate genes for involvment in memory consolidation.

18. A method for the identification of mammalian genes involved in memory consolidation comprising:

(i) generating a first library of nucleic acid probes representative of genes expressed in animals having undergone memory consolidation; (ii) generating a second library of nucleic acid probes representative of genes expressed in animals having at least partial dismption of fomix-mediated afferant signalling to the hippocampus, said dismption affecting memory consolidation having dismption of the afferant signal to the hippocampus (iii) identifying genes that up-or downregulated in the first library of nucleic acids relative to the second library of nucleic acids.

19. The method of claim 18, wherein the first and second nucleic acid libraries are derived from hippocampal tissue.

20. The method of claim 19, further comprising a step of detecting the level of activity of a product encoded by a gene that is identified as being up- or downregulated in the first library of nucleic acids.

21. A method of identifying an agent which promotes memory consolidation in a mammal having undergone dismption of the afferant signal to the hippocampus comprising: (i) subjecting the mammal to a learning and memory test to provide a learned behavior in the mammal;

(ii) administering to the mammal the agent to be assessed;

(iii) determining the extent of expression of a gene identified according to the method of claim 18; and (iv) comparing the expression of the gene with its level of expression in a control mammal.

22. A method for identifying an agent which modulates memory consolidation, comprising:

(i) providing a reaction system for detecting the activity of a product encoded by a gene that is identified as being up- or downregulated in the first library of nucleic acids of claim 18;

(ii) contacting said system with a test compound; and

(iiii) determining if the test compound alters the activity of the gene product.

23. A method for identifying an agent which modulates memory consolidation, comprising,

(i) providing a reaction system for detecting the level of expression of a gene that is identified as being up- or downregulated in the first library of nucleic acids of claim 18;

(ii) contacting said system with a test compound; and

(iii) determining if the test compound alters the level of expression of the gene.

24. A pharmaceutical preparation comprising, a compound identify in the assay of claim 18, 22 or 23, formulated in a pharmaceutically acceptable excipient.

25. A method for enhancing memory consolidation in an animal, or otherwise enhancing the functional performance of CNS neurons, comprising administering to an animal a pharmaceutical preparation of claim 11 or 24.

26. A method for augmenting learning and memory, or otherwise enhancing the functional performance of CNS neurons, comprising administering to an animal a pharmaceutical preparation of claim 11 or 24.

27. The method of claim 25 or 26, further comprising administering, conjointly with the pharmaceutical preparation, one or more of a neuronal growth factor, a neuronal survival factor, and a neuronal tropic factor.

28. The method of claim 25 or 26, further comprising administering, conjointly with the pharmaceutical preparation, an agent that activates CREB-dependent transcription in an amount sufficient to produce a memory enhancing effect.

29. The method of claim 28, wherein the CREB activating agent is a cAMP elevating agent.

30. The method of claim 29, wherein at least one cAMP agonist activates adenylate cyclase.

31. The method of claim 28, wherein the CREB activating agent is a cAMP analog.

32. The method of claim 28, wherein the CREB activating agent is a cAMP phosphodiesterase inhibitor.

33. A method for assessing a patient for learning and/or memory functional performance including a step of detecting the level of CREB phosphorylation in the patient's hippocampus.

34. The method of 33, wherein the level of CREB phosphorylation in the patient's hippocampus is detected by non-invasive spectroscopy, such as Magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS), cerebral computed tomography (CCT), in vivo nmr (e.g., ³¹P NMR), or positron emission tomography (PET) imaging.

35. A method for assessing a patient for learning and/or memory functional performance including a step of detecting the expression of one or more genes identified according to claim 18, or the level of activity of the gene products thereof, in the patient's hippocampus.

36. A method of identifying an agent which modulates memory consolidation in a mammal, comprising (i) providing a non-human mammal which has been manipulated to create at least partial disruption of fornix-mediated afferant signalling to the hippocampus, said disruption affecting memory consolidation,

(ii) conditioning the mammal with a learning or memory regimen which results in learned behavior in the mammal in the absence of the fomix lesion; (iii) ascertaining the effect of one or neurotransmitters, or agonist or antagonist thereof, on the the learned behavior of the mammal; wherein a change in learned behavior, relative to the absence of the neurotransmitter(s), indicates that the neurotransmitter effects memory consolidation.

37. A method of identifying a conjoint therapy which enhances memory consolidation in a mammal, comprising (i) providing a non-human mammal which has been manipulated to create disruption of fomix-mediated afferant signalling to the hippocampus,

(ii) conditioning the mammal with a learning or memory regimen which results in learned behavior in the mammal in the absence of the fomix lesion; (iii) conjointly administering two or more neurotransmitters, or agonists or antagonists thereof, and

(iv) ascertaining the effect of said conjointly administered neurotransmitters on the learned behavior of the mammal; wherein an increase in learned behavior, relative to the absence of the conjointly administered neurotransmitters, indicates that the conjointly administered neurotransmitters enhance memory consolidation.

38. A method for enhancing memory consolidation in an animal, comprising administering to the animal one or more neurotransmitters, or agonists or antagonists thereof, produced by fomix-mediated afferant signalling to the hippocampus affecting memory consolidation.

39. The method of claim 38, comprising administered two or more neurotransmitters, or agonists or antagonists thereof, produced by fomix-mediated afferant signalling to the hippocampus affecting memory consolidation.

40. The method of claim 38 or 39, wherein at least one of the neurotransmitters is an agonist of a neurotransmiter which enhances memory consolidation.

41. The method of claim 38 or 39, wherein at least one of the neurotransmitters is an antagonist of a neurotransmiter which inhibits memory consolidation.

42. A pharmaceutical preparation comprising two or more neurotransmitters, or agonists or antagonists thereof, produced by fomix-mediated afferant signalling to the hippocampus, which neurotransmitters are provided in an amount sufficient to affect memory consolidation in a mammal.

43. A kit including pharmaceutical preparations of two or more neurotransmitters, or agonists or antagonists thereof, produced by fomix-mediated afferant signalling to the hippocampus, which neurotransmitters are provided in an amount sufficient to affect memory consolidation in a mammal.