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  Pharmaceutical Patents  

 

Title:  Method for assessing the effect of a drug on long term memory formation
United States Patent: 
7,572,434
Issued: 
August 11, 2009

Inventors:
 Tully; Timothy P. (Cold Spring Harbor, NY), Yin; Jerry Chi-Ping (Madison, WI)
Assignee:
  Cold Spring Harbor Laboratory (Cold Spring Harbor, NY)
Appl. No.:
 11/066,125
Filed:
 February 25, 2005


 

Executive MBA in Pharmaceutical Management, U. Colorado


Abstract

Methods of assessing the effect of a drug on long term memory and for screening a pharmaceutical agent for its ability to modulate long term memory are disclosed.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention is based on Applicants' discovery of the dCREB1 and dCREB2 genes. The present invention is further based on Applicants' discovery that the Drosophila CREB2 gene codes for proteins of opposite functions. One isoform (e.g., dCREB2-a) encodes a cyclic 3',5'-adenosine monophosphate (cAMP)-responsive transcriptional activator. Another isoform (e.g., dCREB2-b) codes for an antagonist which blocks the activity of the activator.

When the blocking form is placed under the control of the heat-shock promoter, and transgenic flies are made, a brief shift in temperature induces the synthesis of the blocker in the transgenic fly. This induction of the blocker (also referred to herein as the repressor) specifically disrupts long-term, protein synthesis dependent memory of an odor-avoidance behavioral paradigm.

As a result of Applicants' discovery, a method is herein provided to regulate long term memory in an animal. The method of regulating long term memory described herein comprises inducing expression of a dCREB2 gene or a fragment thereof in the animal.

The dCREB2 gene encodes several isoforms. Examples of an isoform encoded by the dCREB2 gene are dCREB2-a, dCREB2-b, dCREB2-c, dCREB2-d, dCREB2-q, dCREB2-r and dCREB2-s.

The isoforms encoded by the dCREB2 gene include cAMP-responsive activator isoforms and antagonistic blocker (or repressor) isoforms of the activator isoforms. Cyclic AMP responsive activator isoforms can function as a cAMP-responsive activator of transcription. Antagonistic repressors can act as a blocker of activators. An example of a cAMP-responsive activator isoform is dCREB2-a. An example of an antagonistic repressor (or blocker) isoform is dCREB2-b. The terms blocker and repressor are used interchangeably herein.

In one embodiment of the invention, the dCREB-2 gene encodes a cAMP-responsive activator isoform and inducing said gene results in the potentiation of long term memory.

Alternatively, inducing the dCREB2 gene encoding a cAMP-responsive activator isoform activates the production of a protein which is necessary for the formation of long term memory.

In another embodiment of the invention, the dCREB2 gene encodes a repressor isoform and inducing said gene results in the blocking of long term memory.

A further embodiment of the invention relates to a method of regulating long term memory in an animal comprising inducing repressor and activator isoforms of dCREB2 wherein long term memory is potentiated in the animal when the net amount of functional activator (.DELTA.C) is greater than zero.

The invention also relates to a method of identifying a substance capable of affecting long term memory in an animal comprising the determination that said substance alters the induction or activity of repressor and activator isoforms of dCREB2 from normal in the animal.

As referred to herein, an activator isoform includes dCREB2-a and functional fragments thereof and a repressor isoform includes dCREB2-b and functional fragments thereof.

Other embodiments of the invention relate to a method of enhancing long term memory formation in an animal comprising increasing the level of activator homodimer from normal, decreasing the level of activator-repressor heterodimer from normal, or decreasing the level of repressor homodimer from normal in the animal.

Still another embodiment of the invention relates to a method of identifying a substance capable of affecting long term memory in an animal comprising the determination that said substance alters activator homodimer, activator-repressor heterodimer and/or repressor homodimer formation from normal in the animal.

As referred to herein, an activator homodimer includes the dCREB2a homodimer, an activator-repressor heterodimer includes the dCREB2a-dCREB2b heterodimer, and a repressor homodimer includes the dCREB2b homodimer.

A further embodiment of the invention relates to isolated DNA encoding a cAMP responsive transcriptional activator. Such a cAMP responsive transcriptional activator can be encoded by a Drosophila dCREB2 gene or by homologues or functional fragments thereof. For example, a cAMP responsive transcriptional activator can be encoded by the dCREB2 gene which codes for dCREB2-a or by a gene encoded by the sequences presented herein.

Still another embodiment of the invention relates to isolated DNA encoding an antagonist of cAMP-inducible transcription. Such an antagonist of cAMP-inducible transcription can be encoded by a Drosophila dCREB2 gene or by homologues or functional fragments thereof. For example, an antagonist of cAMP-inducible transcription can be encoded by the dCREB2 gene which codes for dCREB2-b.

Another embodiment of the invention relates to isolated DNA (SEQ ID NO.: 25) which encodes a Drosophila dCREB2 gene or functional fragments thereof.

A further embodiment of the invention relates to isolated DNA encoding an enhancer-specific activator. Such an enhancer-specific activator can be encoded by a Drosophila dCREB1 gene or by homologues or functional fragments thereof.

Another embodiment of the invention relates to isolated DNA encoding a nitric oxide synthase of Drosophila (DNOS). Such DNA can encode a DNOS of neuronal locus. The DNOS encoded can contain, for example, putative heme, calmodulin, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate, in its reduced form, (NADPH) binding site domains.

A further embodiment of the invention relates to a method for assessing the effect of a drug on long term memory formation comprising administering the drug to Drosophila, subjecting the Drosophila to classical conditioning to at least one odorant and electrical shock, and assessing the performance index of the classical conditioning, wherein the effect of the drug occurs when it alters the performance index from normal. The drug can affect long term memory formation by, for example, altering the induction or activity of repressor and activator isoforms of dCREB2.

A still further embodiment of the invention relates to the assessment that an animal will have an enhanced or, alternatively, a diminished capability of possessing long term memory. This assessment can be performed by determining the amount of cAMP-responsive activator isoforms, cAMP-responsive repressor or blocker isoforms, or dimers of these isoforms that are present in the animal, where these isoforms are encoded by the CREB2 or a homologous gene. Enhanced capability of possessing long term memory will be more likely as the amount of activator exceeds the amount of repressor, i.e. in direct proportion to the size of the net amount of functional activator (.DELTA.C) when this quantity is greater than zero. Conversely, diminished capability of processing long term memory will be more likely as the amount of repressor exceeds the amount of activator, i.e. in direct proportion to the size of the net amount of functional activator (.DELTA.C) when this quantity is less than zero.

Another embodiment of the invention relates to a screening assay of pharmaceutical agents as enhancers of long term memory or as obstructors of long term memory in animals. The screening assay is performed by determining the change in the amount of cAMP-responsive activator isoforms, cAMP-responsive repressor or blocker isoforms, or dimers of these isoforms that is present in an animal or, more preferably, in a cell culture system or in Drosophila when the pharmaceutical agent is present, in comparison to when the pharmaceutical agent is not present, where these isoforms are encoded by the CREB2 or a homologous gene. Enhancers of long term memory cause a net increase in the amount of activator isoforms relative to the amount of repressor isoforms, i.e. an increase in the net amount of functional activator (.DELTA.C). Obstructors of long term memory cause a net decrease in the amount of activator isoforms relative to the amount of repressor isoforms, i.e. a decrease in the net amount of functional activator (.DELTA.C). The pharmaceutical agent can cause these changes by acting, for example, to alter the expression (transcription or translation) of the respective activator and/or repressor isoforms from the CREB2 or a homologous gene, to alter the formation of activator homodimers, activator-repressor heterodimers and/or repressor homodimers from the expressed isoforms, or to alter the interaction of one or more of these isoform or dimer types at their molecular targets. The long term memory activator isoform/repressor isoform system herein disclosed provides a unique platform for conducting such screening assays.

A further embodiment of the invention relates to an assay of pharmaceutical agents for their property as facilitators or hinderers of long term memory in animals. The assay is performed by administering the pharmaceutical agent to Drosophila prior to subjecting the Drosophila to a Pavlovian olfactory learning regimen. This regimen assesses the long term memory capabilities of the Drosophila by subjecting the flies to a massed and/or a spaced training schedule. Transgenic lines of these flies containing altered dCREB2 genes can be used to further elucidate the long term memory facilitation or hindering property of the pharmaceutical agent. The assay provides data regarding the acquisition of long term memory by the Drosophila after exposure to the pharmaceutical agent. These data are compared to long term memory acquisition data from Drosophila that have not been exposed to the pharmaceutical agent. If the exposed flies display faster or better retained long term memory acquisition than the unexposed flies, the pharmaceutical agent can be considered to be a facilitator of long term memory. Conversely, if the exposed flies display slower or less retained long term memory acquisition than the unexposed flies, the pharmaceutical agent can be considered to be a hinderer of long term memory. Since the genetic locus for this long term memory assay in Drosophila resides in the dCREB2 gene, the results from this assay can be directly applied to other animals that have homologous genetic loci (CREB2 or CREM genes).

DETAILED DESCRIPTION OF THE INVENTION

Applicants have cloned and characterized two genes, designated dCREB2 and dCREB1, isolated through a DNA-binding expression screen of a Drosophila head cDNA library in which a probe containing three cAMP-responsive element (CRE) sites was used.

The dCREB2 gene codes for the first known cAMP-dependent protein kinase (PKA) responsive CREB/ATF transcriptional activator in Drosophila. A protein data base search showed mammalian CREB, CREM and ATF-1 gene products as homologous to dCREB2. For these reasons, dCREB2 is considered to be a member, not only of the CREB/ATF family, but of the specific cAMP-responsive CREB/CREM/ATF-1 subfamily. It is reasonable to expect that dCREB2 is involved in Drosophila processes which are analogous to those which are thought to depend on cAMP-responsive transcriptional activation in other animal systems.

Applicants have shown that the dCREB2 transcript undergoes alternative splicing. Splice products of dCREB2 were found to fall into two broad categories: one class of transcripts (dCREB2-a, -b, -c, -d) which employs alternative splicing of exons 2, 4 and 6 to produce isoforms whose protein products all have the bZIP domains attached to different versions of the activation domain and a second class of transcripts (dCREB2-q, -r, -s) which have splice sites which result in in-frame stop codons at various positions upstream of the bZIP domain. These all predict truncated activation domains without dimerization or DNA binary activity.

dCREB2-a, -b, -c and -d are splice forms that predict variants of the activation domain attached to a common basic region-leucine zipper. These alternative splice forms result in seemingly minor changes in the size and spacing of parts of the activation domain. Nevertheless, alternative splicing of the activation domain has profound effects on the functional properties of dCREB2 products. Isoform dCREB2-a produces a PKA-responsive transcriptional activator in cell culture, whereas dCREB2-b, lacking exons 2 and 6, produces a specific antagonist. This dCREB2 splicing pattern (and its functional consequences) is virtually identical to that seen in the CREM gene. Similarly located, alternatively-spliced exons in the CREM gene determine whether a particular isoform is an activator or an antagonist (deGroot, R. P. and P. Sassone-Corsi, Mol. Endocrinol., 7: 145-153 (1993); Foulkes, N. S. et al., Nature, 355: 80-84 (1992)).

The ability of the phosphorylation domain (KID domain) to activate in trans other constitutive transcription factors which are bound nearby could potentially transform a CREM antagonist (which contains the KID domain but is lacking an exon needed for activation) into a cAMP-responsive activator. Since the modular organization of these molecules has been conserved, dCREB2-d could have this property.

In contrast to the dCREB2 splicing variants that encode isoforms with a basic region-leucine zipper domain, the dCREB2-q, -r and -s splice forms incorporate in-frame stop codons whose predicted protein products are truncated before the bZIP region. Isoforms of this type have been identified among the products of the CREB gene (deGroot, R. P. and P. Sassone-Corsi, Mol. Endocrinol., 7: 145-153 (1993); Ruppert, S. et al., EMBO J, 11: 1503-1512 (1992)) but not the CREM gene. The function of these truncated CREB molecules is not known, but at least one such CREB mRNA is cyclically regulated in rat spermatogenesis (Waeber, G. et al., Mol. Endocrinol., 5: 1418-1430 (1991)).

So far, dCREB2 is the only cAMP-responsive CREB transcription factor isolated from Drosophila. Other Drosophila CREB molecules, BBF-2/dCREB-A (Abel, T. et al., Genes Dev., 6: 466-488 (1992); Smolik, S. M. et al., Mol. Cell Biol., 12: 4123-4131 (1992)), dCREB-B (Usui, T. et al., DNA and Cell Biology, 12(7): 589-595 (1993)) and dCREB1, have less homology to mammalian CREB and CREM. It may be that dCREB2 subsumes functions of both the CREB and CREM genes in Drosophila. The mammalian CREB and CREM genes are remarkably similar to one another in several respects. It has been suggested that CREB and CREM are the product of a gene duplication event (Liu, F. et al., J. Biol. Chem., 268: 6714-6720 (1993); Riabowol, K. T. et al., Cold Spring Harbor Symp. Quant. Biol., 1: 85-90 (1988)). dCREB2 has a striking degree of amino acid sequence similarity to the CREB and CREM genes in the bZIP domain. Moreover, comparison of alternative splicing patterns among CREB, CREM and dCREB2 indicates that dCREB2 generates mRNA splicing isoforms similar to exclusive products of both CREB and CREM. Taken together, the sequence information and the splicing organization suggest that dCREB2 is an ancestor of both the mammalian CREB and CREM genes.

As discussed further herein, one phenomenon in which dCREB2 might act with enduring consequences is in long-term memory. This possibility is a particularly tempting one because recent work in Aplysia indicates that a CREB factor is likely to function in long-term facilitation by inducing an "immediate early" gene (Alberini, C. M. et al., Cell, 76: 1099-1114 (1994); Dash, P. K., Nature, 345: 718-721 (1990)). Recent experiments with a conditionally-expressed dCREB2-b transgene indicate that it has specific effects on long-term memory in Drosophila.

The product of the second gene described herein, dCREB1, also appears to be a member of the CREB/ATF family. Gel-retardation assays indicate that it binds specifically to CREs. It has a basic region and an adjacent leucine zipper at its carboxyl end, but this domain shows limited amino acid sequence similarity to other CREB/ATF genes. The presumed transcriptional activation domain of dCREB1 is of the acid-rich variety. Furthermore, it has no consensus phosphorylation site for PKA. dCREB1 can mediate transcriptional activation from CRE-containing reporters in the Drosophila L2 cell line, but this activation is not dependent on PKA.

A recurrent finding from work on the biology of learning and memory is the central involvement of the cAMP signal transduction pathway. In Aplysia, the cAMP second-messenger system is critically involved in neural events underlying both associative and non-associative modulation of a behavioral reflex (Kandel, E. R. and J. H. Schwartz, Science, 218: 433-443 (1982); Kandel, E. R., et al., In Synaptic Function, Edelmann, G. M., et al. (Eds.), John Wiley and Sons, New York (1987); Byrne, J. H., et al., In Advances in Second Messenger and Phosphoprotein Research, Shenolikar, S. and A. C. Nairn (Eds.), Raven Press, New York, pp. 47-107 (1993)). In Drosophila, two mutants, dunce and rutabaga, were isolated in a behavioral screen for defects in associative learning and are lesioned in genes directly involved in cAMP metabolism (Quinn, W. G., et al., Proc. Natl. Acad. Sci. USA, 71: 708-712 (1974); Dudai, Y., et al., Proc. Natl. Acad. Sci., USA 73: 1684-1688 (1976); Byers, D. et al., Nature, 289: 79-81 (1981); Livingstone, M. S., et al., Cell, 37: 205-215 (1984); Chen, C. N. et al., Proc. Natl. Acad. Sci. USA, 83: 9313-9317 (1986); Levin, L. R., et al., Cell, 68: 479-489 (1992)). These latter observations were extended with a reverse-genetic approach using inducible transgenes expressing peptide inhibitors of cAMP-dependent protein kinase (PKA) and with analyses of mutants in the PKA catalytic subunit (Drain, P. et al., Neuron, 6: 71-82 (1991); Skoulakis, E. M., et al., Neuron, 11: 197-208 (1993)). Recent work on mammalian long-term potentiation (LTP) also has indicated a role for cAMP in synaptic plasticity (Frey, U., et al., Science, 260: 1661-1664 (1993); Huang, Y. Y. and E. R. Kandel, In Learning and Memory, vol. 1, pp. 74-82, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1994)).

The formation of long-lasting memory in animals and of long-term facilitation in Aplysia can be disrupted by drugs that interfere with transcription or translation (Agranoff, B. W. et al., Brain Res., 1: 303-309 (1966); Barondes, S. H. and H. D. Cohen, Nature, 218: 271-273 (1968); Davis, H. P. and L. R. Squire, Psychol. Bull., 96: 518-559 (1984); Rosenzweig, M. R. and E. L. Bennett, In Neurobiology of Learning and Memory, Lynch, G., et al. (Eds.), The Guilford Press, New York, pp. 263-288, (1984); Montarolo, P. G., et al., Science, 234: 1249-1254 (1986)). This suggests that memory consolidation requires de novo gene expression. Considered along with the involvement of the cAMP second-messenger pathway, this requirement for newly synthesized gene products suggests a role for cAMP-dependent gene expression in long-term memory (LTM) formation.

In mammals, a subset of genes from the CREB/ATF family are known to mediate cAMP-responsive transcription (Habener, J. F., Mol. Endocrinol., 4: 1087-1094 (1990); deGroot, R. P. and P. Sassone-Corsi, Mol. Endocrinol., 7: 145-153 (1993)). CREBs are members of the basic region-leucine zipper transcription factor superfamily; (Landschulz, W. H. et al., Science, 240: 1759-1764 (1988)). The leucine zipper domain mediates selective homo- and hetero-dimer formation among family members (Hai, T. Y. et al., Genes & Dev., 3: 2083-2090 (1989); Hai, T. and T. Curran, Proc. Natl. Acad. Sci. USA, 88: 3720-3724 (1991)). CREB dimers bind to a conserved enhancer element (CRE) found in the upstream control region of many cAMP-responsive mammalian genes (Yamamoto, K. K., et al., Nature, 334: 494-498 (1988)). Some CREBs become transcriptional activators when specifically phosphorylated by PKA (Gonzalez, G. A. and M. R. Montminy, Cell, 59: 675-680 (1989); Foulkes, N. S. et al., Nature, 355: 80-84 (1992)), while others, isoforms from the CREM gene, are functional antagonists of these PKA-responsive activators (Foulkes, N. S. et al., Cell, 64: 739-749 (1991); Foulkes, N. and P. Sassone-Corsi, Cell, 68: 411-414 (1992)).

Work in Aplysia has shown that cAMP-responsive transcription is involved in long-term synaptic plasticity (Schacher, S. et al., Science, 240: 1667-1669 (1988); Dash, P. K., Nature, 345: 718-721 (1990)). A primary neuronal co-culture system has been used to study facilitation of synaptic transmission between sensory and motor neurons comprising the monosynaptic component of the Aplysia gill-withdrawal reflex. Injection of oligonucleotides containing CRE sites into the nucleus of the sensory neuron specifically blocked long-term facilitation (Dash, P. K., Nature, 345: 718-721 (1990)). This result suggests that titration of CREB activity might disrupt long-term synaptic plasticity.

Described herein is the cloning and characterization of a Drosophila CREB gene, dCREB2. This gene produces several isoforms that share overall structural homology and nearly complete amino acid identity in the basic region-leucine zipper with mammalian CREBs. The dCREB2-a isoform is a PKA-responsive transcriptional activator whereas the dCREB2-b product blocks PKA-responsive transcription by dCREB2-a in cell culture. These molecules with opposing activities are similar in function to isoforms of the mammalian CREM gene (Foulkes, N. S. et al., Cell, 64: 739-749 (1991); Foulkes, N. and P. Sassone-Corsi, Cell, 68: 411-414 (1992); Foulkes, N. S. et al., Nature, 355: 80-84 (1992)). The numerous similarities in sequence and function between dCREB2 and mammalian CREBs suggest that cAMP-responsive transcription is evolutionarily conserved.

Genetic studies of memory formation in Drosophila have revealed that the formation of a protein synthesis-dependent long-term memory (LTM) requires multiple training sessions with a rest interval between them. As discussed further herein, this LTM is blocked specifically by induced expression of a repressor isoform of the cAMP-responsive transcription factor CREB. Also as discussed further herein, LTM information is enhanced after induced expression of an activator form of CREB. Maximum LTM is achieved after just one training session.

To investigate the role of CREBs in long-term memory (LTM) formation in Drosophila, dominant-negative transgenic lines which express dCREB2-b under the control of a heat-shock promoter (hs-dCREB2-b) were generated. Groups of flies, which had been heat-shock induced or left uninduced, were tested for memory retention after Pavlovian olfactory learning. This acute induction regimen minimized potential complications from inappropriate expression of dCREB2-b during development and allowed a clear assessment of the effect of hs-dCREB2-b induction on memory formation.

In Drosophila, consolidated memory after olfactory learning is composed of two genetically distinct components: anesthesia-resistant memory (ARM) and long-term memory (LTM). ARM decays to zero within four days after training, and formation of ARM is insensitive to the protein synthesis inhibitor cycloheximide (CXM) but is disrupted by the radish mutation (Folkers, E., et al., Proc. Natl. Acad. Sci. USA, 90: 8123-8127 (1993)). In contrast, LTM shows essentially no decay over at least seven days, its formation is cycloheximide-sensitive and it is not disrupted by radish. Two different training protocols involving massed and spaced sessions were employed (Ebbinghaus, H., Uber das Gedachtnis, Dover, N.Y.(1885); Baddeley, A. D., The Psychology of Memory, Basic Books, N.Y. (1976)) to dissect memory formation. The massed training procedure consists of ten consecutive training cycles with no rest interval between them, while the spaced training protocol consists of the same number of sessions but with a 15-minute rest between each. Their genetic dissection revealed that the massed protocol produced only ARM, while the spaced protocol produced memory retention composed of both ARM and LTM.

The behavioral results show that formation of LTM is completely blocked by induced expression of hs-dCREB2-b. This effect is remarkable in its behavioral specificity. ARM, a form of consolidated memory genetically distinguishable from LTM, but co-existing with it one-day after spaced training, was not affected. Learning and peripheral behaviors likewise were normal. Thus, the effect of the induced hs-dCREB2-b transgene is specific to LTM.

Induction of the mutant blocker did not affect LTM. This result, together with the molecular data which showed that induction of the wild-type blocker did not have widespread effects on transcription, suggests that the blocker is reasonably specific at the molecular level when it specifically blocks LTM. The wild-type blocker may disrupt cAMP-dependent transcription in vivo, since it can block cAMP-responsive transcription in cell culture. It is reasonable to infer that dimerization is necessary for blocker function and that the wild-type blocker could interfere with cAMP-responsive transcription either by forming heterodimers with dCREB2-a, the activator, or by forming homodimers and competing for DNA binding with homodimers of dCREB2-a. Thus, activators and repressors may form homodimers or heterodimers. It is reasonable to infer that long term memory is enhanced when the level of activator homodimer is increased from normal and/or when the level of activator-repressor heterodimer is decreased from normal and/or when the level of repressor homodimer is decreased from normal. In any case, the molecular target(s) of dCREB2-b are likely to be interesting because of the behavioral specificity of the block of LTM.

In Drosophila, consolidation of memory into long-lasting forms is subject to disruption by various agents. A single-gene mutation radish and the pharmacological agent CXM were used to show that long-lasting memory in flies is dissectable into two components, a CXM-insensitive ARM, which is disrupted by radish, and a CXM-sensitive LTM, which is normal in radish mutants. As described herein, CREB-family members are likely to be involved in the CXM-sensitve, LTM branch of memory consolidation. The results described herein, taken together with the showing that long-term memory is dissectable into a CXM-insentive ARM and a CXM-sensitive LTM, show that only one functional component of consolidated memory after olfactory learning lasts longer than four days, requires de novo protein synthesis and involves CREB-family members.

Based on work in Aplysia, a model has been proposed to describe the molecular mechanism(s) underlying the transition from short-term, protein synthesis-independent to long-term, protein synthesis-dependent synaptic plasticity (Alberini, C. M. et al., Cell, 76: 1099-1114 (1994)). The present work in Drosophila on long-term memory extends this model to the whole organism. Important molecular aspects of this transition seem to involve migration of the catalytic subunit of PKA into the nucleus (Backsai, B. J. et al., Science, 260: 222-226 (1993)) and subsequent phosphorylation and activation of CREB-family members (Dash, P. K., Nature, 345: 718-721 (1990); Kaang, B. K., et al., Neuron, 10: 427-435 (1993)). In flies, it is likely that the endogenous dCREB2-a isoform is one of these nuclear targets. Activated dCREB2-a molecules then might transcribe other target genes, including the immediate early genes--as is apparently the case in Aplysia. (Alberini, C. M. et al., Cell, 76: 1099-1114 (1994)).

It is remarkable that the cAMP signal transduction pathway, including its nuclear components, seem to be required for memory-related functions in each of these species and behavioral tasks. Taken together with cellular analyses of a long-lasting form of LTP in hippocampal slices (Frey, U., et al., Science, 260: 1661-1664 (1993); Huang, Y. Y. and E. R. Kandel, In Learning and Memory, vol. 1, pp. 74-82, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1994)), the emerging picture is that cAMP-responsive transcription is a conserved molecular switch involved in the consolidation of short-term memory to long-term memory. Thus, it is reasonable to infer that differential regulation of CREB isoforms serves as a molecular switch for the formation of long term memory.

A universal property of memory formation is that spaced training (repeated training sessions with a rest interval between them) produces stronger, longer-lasting memory than massed training (the same number of training sessions with no rest interval) (Ebbinghaus, H., Uber das Gedachtnis, Dover, N.Y. (1885); Hintzman, D. L., In Theories in Cognitive Psychology: The Loyola Symposium, R. L. Solso (Ed.), pp. 77-99, Lawrence Erlbaum Assoc., Hillsdale, N.J. (1974); Carew, T. J., et al., Science, 175: 451-454 (1972); Frost, W. N., et al., Proc. Natl. Acad. Sci. USA, 82: 8266-8269 (1985)). This phenomenon also exists in fruit flies for a conditioned odor avoidance response (Tully, T. and W. G. Quinn, J. Comp. Physiol. 157: 263-277 (1985)). Genetic dissection of this long-lasting memory has revealed, however, an important difference between massed and spaced training. Spaced training produces two functionally independent forms of consolidated memory, ARM and LTM, while massed training produces only ARM.

As described herein, ARM and LTM differ primarily in their requirement for protein synthesis during induction. ARM is not affected when flies are fed the protein synthesis inhibitor cycloheximide (CXM) immediately before or after training, while LTM is completely blocked under the same feeding conditions. ARM in normal flies also decays away within four days after training, while LTM shows no decay for at least seven days. Thus, protein synthesis is required to induce LTM, but LTM is maintained indefinitely once formed. These latter properties of LTM have been observed throughout the animal kingdom (Davis, H. P. and L. R. Squire, Psychol. Bull., 96: 518-559 (1984); Castellucci, V. F., et al., J. Neurobiol., 20: 1-9 (1989); Erber, J., J. Comp.Physiol.Psychol., 90: 41-46 (1976); Jaffe, K., Physiol.Behav., 25: 367-371 (1980)). The emerging neurobiological interpretation is that formation of LTM involves protein synthesis-dependent structural changes at relevant synapses (Greenough, W. T., TINS, 7: 229-283 (1984); Buonomano, D. V. and J. H. Byrne, Science, 249: 420-423 (1990); Nazif, F. A., et al., Brain Res., 539: 324-327 (1991); Stewart, M. G., In Neural and Behavioural Plasticity: The Use of the Domestic Chick As A Model, R. J. Andrew (Ed.), pp. 305-328, Oxford, Oxford (1991); Bailey, C. H. and E. R. Kandel, Sem. Neurosci., 6:35-44 (1994)). The modern molecular view is that regulation of gene expression underlies this protein synthesis-dependent effect (Goelet, P. et al., Nature, 322: 419-422 (1986); Gall, C. M. and J. C. Lauterborn, In Memory:Organization and Locus of Change, L. R. Squire, et al., (Eds.) pp. 301-329 (1991); Armstrong, R. C. and M. R. Montminy, Annu.Rev.Neurosci., 16: 17-29 (1993)).

Why is spaced training required to induce LTM? The massed and spaced procedures both entail ten training sessions; consequently, flies receive equivalent exposure to the relevant stimuli (one odor temporally paired with electric shock and a second odor presented without shock). The only procedural difference between massed and spaced training is the rest interval between each training session. The absence of a rest interval between sessions during massed training does not appear to disrupt the memory formation process. The level of initial learning assayed immediately after massed training is similar to that after spaced training. In addition, ARM levels are similar after both training procedures. Thus, the presence of a rest interval during spaced training seems crucial to the induction of LTM.

To investigate the temporal kinetics of this rest interval in relation to the formation of LTM (FIGS. 13A and 13B, see Original Patent), it was first established that the usual ten sessions of spaced training produced maximal 7-day memory retention (7-day retention is composed solely of LTM, since ARM decays to zero within four days.

FIG. 13A (see Original Patent) shows that 15 or 20 training sessions did not improve memory retention. Thus, ten spaced training sessions produces maximal, asymptotic levels of LTM.

LTM as a function of the length of the rest interval between 10 spaced training sessions was then assessed. FIG. 13B (see Original Patent) reveals a continuous increase in LTM from a O-min rest interval (massed training) to a 10-minute rest interval, at which time LTM levels reach maximum. Longer rest intervals yielded similar memory scores. These observations of LTM formation suggest an underlying biological process, which changes quantitatively during the rest interval between sessions and which accumulates over repeated training sessions.

In transgenic flies, the formation of LTM, but not ARM or any other aspect of learning or memory, is disrupted by induced expression of a repressor form of the cAMP-responsive transcription factor CREB (Example 4). Mutating two amino acids in the "leucine zipper" dimerization domain of this CREB repressor was sufficient to prevent the dominant-negative effect on LTM. Thus, indication of LTM is not only protein synthesis-dependent but also is CREB-dependent. Stated more generally, CREB function is involved specifically in a form of a memory that is induced only by spaced training. This observation was particularly intriguing in light of the molecular nature of CREB.

In Drosophila, transcriptional and/or post-translational regulation of dCREB2 yields several mRNA isoforms. Transient transfection assays in mammalian F9 cells have demonstrated that one of these isoforms (CREB2-a) functions as a cAMP-responsive activator of transcription, while a second isoform (CREB2-b) acts as an antagonistic repressor of the activator (Example 1; cf. Habener, J. F., Mol. Endocrinol., 4: 1087-1094 (1990); Foulkes, N. and P. Sassone-Corsi, Cell, 68: 411-414 (1992)). (This repressor isoform was used previously to generate the inducible transgene mentioned above.) The existence of different CREB isoforms with opposing functions suggested an explanation for the requirement of multiple training sessions with a rest interval between them for the formation of LTM.

In its simplest form, this model (Example 7; FIG. 14 (see Original Patent)) supposes that cAMP-dependent protein kinase (PKA), activated during training, induces the synthesis and/or function of both CREB activator and repressor isoforms (cf. Yamamoto, K. K., et al., Nature, 334: 494-498 (1988); Backsai, B. J. et al., Science, 260: 222-226 (1993)). Immediately after training, enough CREB repressor exists to block the ability of CREB activator to induce downstream events. Then, CREB repressor isoforms are inactivated faster than CREB activator isoforms. In this manner, the net amount of functional activator (.DELTA.C=CREB2a-CREB2b) increases during a rest interval and then accumulates over repeated training sessions (with a rest interval) to induce further the downstream targets involved with the formation of LTM (Montarolo, P. G., et al., Science, 234: 1249-1254 (1986); Kaang, B. K., et al., Neuron, 10: 427-435 (1993)).

This model leads to three predictions, which have been confirmed. First, if the functional difference between CREB activator and repressor isoforms is zero (.DELTA.C=0) immediately after one training session, then additional massed training sessions should never yield LTM. FIG. 15A (see Original Patent) shows that 48 massed training sessions, rather than the usual 10, still does not produce any 7-day memory retention. Second, if the amount of CREB repressor is increased experimentally, .DELTA.C will be negative immediately after training (.DELTA.C<0). Then, enough CREB repressor may not decay during a rest interval to free enough CREB activator for induction of LTM. This has been shown to be the case for spaced training (15-min rest interval) after inducing the expression of a hsp-dCREB2-b (repressor) transgene three hours before training (Example 4). Third, if the amount of CREB activator is increased experimentally, .DELTA.C will be positive immediately after training (.DELTA.C>0). This effect, then, should eliminate or reduce the rest interval required to induce LTM. FIG. 15B shows the results from recent experiments in which the expression of a hsp-dCREB2-a (activator) transgene was induced three hours before training. In these transgenic flies, massed training produced maximal LTM. This effect appeared not to arise trivially, since olfactory acuity, shock reactivity (FIG. 15C (see Original Patent)) and initial learning were normal in transgenic flies after heat shock-induction. Thus, the requirement for a rest interval between training sessions to induce LTM specifically was eliminated.

FIG. 15B (see Original Patent) also shows that maximal LTM occurred in induced hsp-dCREB2-a transgenic flies after just one training session. The usual requirement for additional training to form a strong, long-lasting memory was no longer necessary. Thus, induced overexpression of a CREB activator has produced in otherwise normal flies, the functional equivalent of a "photographic" memory. This result indicates that the amount of CREB activator present during training--rather than the amount of activated PKA that reaches CREB in the nucleus, for instance (cf. Backsai, B. J. et al., Science, 260: 222-226 (1993); Kaang, B. K., et al., Neuron, 10: 427-435 (1993); Frank, D. A. and M. E. Greenberg, Cell, 79: 5-8 (1994))--is the rate-limiting step of LTM formation. Taken together, results from these experiments support the notion that the opposing functions of CREB activators and repressors act as a "molecular switch" (cf. Foulkes, N. S. et al., Nature, 355: 80-84 (1992)) to determine the parameters of extended training (number of training sessions and rest interval between them) required to form maximum LTM.

To date, seven different dCREB2 RNA isoforms have been identified, and more are hypothesized to exist. Each may be regulated differentially at transcriptional (Meyer, T. E., et al., Endocrinology, 132: 770-780 (1993)) and/or translation levels before or during LTM formation. In addition, different combinations of CREB isoforms may exist in different (neuronal) cell types. Consequently, many different combinations of activator and repressor molecules are possible. From this perspective, the notions that all activators and repressors are induced during a training session or that all repressors inactivate faster than activators (see above) need not be true. Instead, the model requires only that .DELTA.C (the net function of activators and repressors) is less than or equal to zero immediately after training and increases with time (rest interval).

Theoretically, particular combinations of activator and repressor molecules in the relevant neuron(s) should determine the rest interval and/or number of training sessions necessary to produce maximum LTM for any particular task or species. Thus, the molecular identification and biochemical characterization of each CREB activator and repressor isoform used during LTM formation in fruit flies is the next major step toward establishing the validity of our proposed model. Similar experiments in other species may establish its generality.

CREB certainly is not involved exclusively with LTM. The dCREB2 gene, for instance, is expressed in all fruit fly cells and probably acts to regulate several cellular events (Foulkes, N. S. et al., Nature, 355: 80-84 (1992)).

So, what defines the specificity of its effects on LTM? Specificity most likely resides with the neuronal circuitry involved with a particular learning task. For olfactory learning in fruit flies, for instance, CREB probably is modulated via the cAMP second messenger pathway. Genetic disruptions of other components of this pathway are known to affect olfactory learning and memory (Livingstone, M. S., et al., Cell, 37: 205-215 (1984); Drain, P. et al., Neuron, 6: 71-82 (1991); Levin, L. R., et al., Cell, 68: 479-489 (1992); Skoulakis, E. M., et al., Neuron 11: 197-208 (1993); Qiu, Y. and R. L. Davis, Genes Develop. 7: 1447-1458 (1993)). Presumably, the stimuli used during conditioning (training) stimulate the underlying neuronal circuits. The cAMP pathway is activated in (some) neurons participating in the circuit, and CREB-dependent regulation of gene expression ensues in the "memory cells". This neurobiological perspective potentially will be established in Drosophila by identifying the neurons in which LTM-specific CREB function resides. Experiments using a neuronal co-culture system in Aplysia already have contributed significantly to this issue (Alberini, C. M. et al., Cell, 76: 1099-1114 (1994) and references therein).

The involvement of CREB in memory, or in the structural changes of neurons which underlie memory in vivo, also has been implicated in mollusks (Dash, P. K., Nature, 345: 718-721 (1990); Alberini, C. M. et al., Cell, 76: 1099-1114 (1994)) and in mice (Bourtchuladze, R., et al., Cell, 79: 59-68 (1994)). Ample evidence also exists for the involvement of the cAMP second messenger pathway in associative learning in Aplysia (Kandel, E. R., et al., In Synaptic Function, Edelmann, G. M., et al. (Eds.), John Wiley and Sons, New York (1987); Byrne, J. H., et al., In Advances in Second Messenger and Phosphoprotein Research, Shenolikar, S. and A. C. Nairn (Eds.), Raven Press, New York, pp. 47-107 (1993)) and in rat hippocampal long-term potentiation (LTP), a cellular model of associative learning in vertebrates (Frey, U., et al., Science, 260: 1661-1664 (1993); Huang, Y. Y. and E. R. Kandel, In Learning and Memory, vol. 1, pp. 74-82, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1994)). Finally, cellular and biochemical experiments have suggested that CREB function may be modulated by other second messenger pathways (Dash, P. K., et al., Proc. Natl. Acad. Sci. USA 88: 5061-5065 (1991); Ginty, D. D. et al., Science, 260: 238-241 (1993); deGroot, R. P. and P. Sassone-Corsi, Mol. Endocrinol., 7: 145-153 (1993)). These observation suggest that CREB might act as a molecular switch for LTM in many species and tasks.

Finally, why might the formation of LTM require a molecular switch? Many associative events occur only once in an animal's lifetime. Forming long-term memories of such events would be unnecessary and if not counterproductive. Instead, discrete events experienced repeatedly are worth remembering. In essence, a recurring event comprises a relevant signal above the noise of one-time events. Teleologically, then, the molecular switch may act as an information filter to ensure that only discrete but recurring events are remembered. Such a mechanism would serve efficiently to tailor an individual's behavioral repertoire to its unique environment.

The present invention also relates to isolated DNA having sequences which encode (1) a cyclic 3',5'-adenosine monophosphate (cAMP) responsive transcriptional activator, or a functional fragment thereof, or (2) an antagonist of a cAMP responsive transcriptional activator, or a functional fragment thereof, or (3) both an activator and an antagonist, or functional fragments thereof of both.

The invention relates to isolated DNA having sequences which encode Drosophila dCREB2 isoforms, or functional analogues of a dCREB2 isoform. As referred to herein, a functional analogue of a dCREB2 isoform comprises at least one function characteristic of a Drosophila dCREB2 isoform, such as a cAMP-responsive transcriptional activator function and/or an antagonistic repressor of the cAMP activator function. These functions (i.e., the capacity to mediate PKA-responsive transcription) may be detected by standard assays (e.g., assays which monitor for CREB-dependent activation). For example, assays in F9 cells have been used extensively to study CREB-dependent activation because their endogenous cAMP-responsive system is inactive; (Gonzalez, G. A. et al., Nature, 337: 749-752 (1989); Masson, N. et al., Mol. Cell Biol., 12: 1096-1106 (1992); Masson, N. et al., Nucleic Acids Res., 21: 1163-1169 (1993)).

The invention further relates to isolated DNA having sequences which encode a Drosophila dCREB2 gene or a functional fragment thereof. Isolated DNA meeting these criteria comprise nucleic acids having sequences identical to sequences of naturally occurring Drosophila dCREB2 and portions thereof, or variants of the naturally occurring sequences. Such variants include mutants differing by the addition, deletion or substitution of one or more nucleic acids.

The invention relates to isolated DNA that are characterized by (1) their ability to hybridize to a nucleic acid having the DNA sequence in FIG. 1A (SEQ ID NO.: 1 (see Original Patent)) or its complement, or (2) by their ability to encode a polypeptide of the amino acid sequence in FIG. 1A (SEQ ID NO.: 2) or functional equivalents thereof (i.e., a polypeptide which functions as a cAMP responsive transcriptional activator), or (3) by both characteristics. Isolated nucleic acids meeting these criteria comprise nucleic acids having sequences homologous to sequences of mammalian CREB, CREM and ATF-1 gene products. Isolated nucleic acids meeting these criteria also comprise nucleic acids having sequences identical to sequences of naturally occurring dCREB2 or portions thereof, or variants of the naturally occurring sequences. Such variants include mutants differing by the addition, deletion or substitution of one or more residues, modified nucleic acids in which one or more residues is modified (e.g., DNA or RNA analogs), and mutants comprising one or more modified residues.

Such nucleic acids can be detected and isolated under high stringency conditions or moderate stringency conditions, for example. "High stringency conditions" and "moderate stringency conditions" for nucleic acid hybridizations are explained on pages 2.10.1-2.10.16 (see particularly 2.10.8-11) and pages 6.3.1-6 in Current Protocols in Molecular Biology (Ausubel, F. M. et al., eds, Vol. 1, Suppl. 26, 1991), the teachings of which are incorporated herein by reference. Factors such as probe length, base composition, percent mismatch between the hybridizing sequences, temperature and ionic strength influence the stability of nucleic acid hybrids. Thus, high or moderate stringency conditions can be determined empirically, depending in part upon the characteristics of the known DNA to which other unknown nucleic acids are being compared for homology.

Isolated nucleic acids that are characterized by their ability to hybridize to a nucleic acid having the sequence in FIG. 1A or its complement (e.g., under high or moderate stringency conditions) may further encode a protein or polypeptide which functions as a cAMP responsive transcriptional activator.

The present invention also relates to isolated DNA having sequences which encode an enhancer-specific activator, or a functional fragment thereof.

The invention further relates to isolated DNA having sequences which encode a Drosophila dCREB 1 gene or a functional fragment thereof. Isolated DNA meeting these criteria comprise nucleic acids having sequences identical to sequences of naturally occurring Drosophila dCREB1 and portions thereof, or variants of the naturally occurring sequences. Such variants include mutants differing by the addition, deletion or substitution of one or more nucleic acids.

The invention further relates to isolated DNA that are characterized by (1) their ability to hybridize to a nucleic acid having the DNA sequence in FIG. 5 (SEQ ID NO.: 7 (see Original Patent)) or its complement, or (2) by their ability to encode a polypeptide of the amino acid sequence in FIG. 5 (SEQ ID NO.: 8), or by both characteristics. Isolated DNA meeting these criteria also comprise nucleic acids having sequences identical to sequences of naturally occurring dCREB1 or portions thereof, or variants of the naturally occurring sequences. Such variants include mutants differing by the addition, deletion or substitution of one or more residues, modified nucleic acids in which one or more residues is modified (e.g., DNA or RNA analogs), and mutants comprising one or more modified residues.

Such nucleic acids can be detected and isolated under high stringency conditions or moderate stringency conditions as described above, for example.

Fragments of the isolated DNA which code for polypeptides having a certain function can be identified and isolated by, for example, the method of Jasin, M., et al., U.S. Pat. No. 4,952,501.

Nitric Oxide in Invertebrates: Drosophila dNOS Gene Codes for a Ca.sup.2+/Calmodulin-Dependent Nitric Oxide Synthase.

Nitric oxide (NO) is a gaseous mediator of a wide variety of biological processes in mammalian organisms. Applicants have cloned the Drosophila gene, dNOS, coding for a Ca.sup.2+/calmodulin-dependent nitric oxide synthase (NOS). Presence of a functional NOS gene in Drosophila provides conclusive evidence that invertebrates synthesize NO and presumably use it as a messenger molecule. Furthermore, conservation of an alternative RNA splicing pattern between dNOS and vertebrate neuronal NOS, suggests broader functional homology in biochemistry and/or regulation of NOS.

NO is synthesized by nitric oxide synthases (NOSs) during conversion of L-arginine to L-citrulline (Knowels, R. G., et al., Biochem. J, 298: 249 (1994); Nathan, C., et al., J. Biol. Chem., 269: 13725 (1994); Marletta, M. A., J. Biol. Chem., 268: 12231 (1993)). Biochemical characterization of NOSs has distinguished two general classes: (i) constitutive, dependent on exogenous Ca.sup.2+ and calmodulin and (ii) inducible, independent of exogenous Ca.sup.2+ and calmodulin. Analyses of cDNA clones have identified at least three distinct NOS genes in mammals (Bredt, D. S., et al., Nature, 351: 714-718 (1991); Lamas, S., et al., Proc. Natl. Acad. Sci. USA, 89: 6348-6352 (1992); Lyons, C. R., et al., J. Biol. Chem., 267: 6370 (1992); Lowenstein, C. J., et al., Proc. Natl. Acad. Sci. USA, 89: 6711 (1992); Sessa, W. C., et al., J. Biol. Chem., 267:15274 (1992); Geller, D. A., et al., Proc. Natl. Acad. Sci. USA, 90: 3491 (1993); Xie, Q. et al., Science, 256: 225-228 (1992)) neuronal, endothelial and macrophage, the former two of which are constitutive and the latter of which is inducible. The nomenclature for these different isoforms used here is historical, as it is clear now that one or more isoforms can be present in the same tissues (Dinerman, J. L., et al., Proc. Natl. Acad. Sci. USA, 91: 4214-4218 (1994)).

As a diffusible free-radical gas, NO is a multifunctional messenger affecting many aspects of mammalian physiology [for reviews, see Dawson, T. M., et al., Ann. Neurol., 32: 297 (1992); Nathan, C., FASEB J, 6: 3051 (1992); Moncada, S., et al., N. Eng. J. Med., 329: 2002-2012 (1993); Michel, T., et al., Amer. J. Cardiol., 72: 33C (1993); Schuman, E. M., et al., Annu. Rev. Neurosci., 17: 153-183 (1994)]. NO originally was identified as an endothelium-derived relaxing factor responsible for regulation of vascular tone (Palmer, R. M. J., Nature, 327: 524 (1987); Palmer, R. M. J., et al., Nature, 333: 664 (1988); Ignarro, L. J., et al., Proc. Natl. Acad. Sci. USA, 84: 9265 (1987)) and as a factor involved with macrophage-mediated cytotoxicity (Marletta, M. A., et al., Biochemistry, 21: 8706 (1988); Hibbs, J. B., et al., Biochem. Biophys. Res. Comm., 157: 87 (1989); Steuhr, D. J., et al., J. Exp. Med., 169: 1543 (1989)). Since NO has been implicated in several physiological processes including inhibition of platelet aggregation, promotion of inflammation, inhibition of lymphocyte proliferation and regulation of microcirculation in kidney (Radomski, M., et al., Proc. Natl. Acad. Sci. USA, 87: 5193 (1990); Albina, J. E., J. Immunol., 147: 144 (1991); Katz, R., Am. J. Physiol., 261: F360 (1992); lalenti, A., et al., Eur. J. Pharmacol., 211: 177 (1992)). More recently, NO also has been shown to play a role in cell-cell interactions in mammalian central and peripheral nervous systems--in regulating neurotransmitter release, modulation of NMDA receptor-channel functions, neurotoxicity, nonadrenergic noncholonergic intestinal relaxation (Uemura, Y., et al., Ann. Neurol., 27: 620-625 (1990)) and activity-dependent regulation of neuronal gene expression (Uemura, Y., et al., Ann. Neurol., 27: 620 (1990); Dawson, V. L., et al., Proc. Natl. Acad. Sci. USA, 88: 6368 (1991); Lei, S. Z., et al., Neuron, 8: 1087 (1992); Prast, H., et al., Eur. J. Pharmacol., 216: 139 (1992); Peunova, N., Nature, 364: 450 (1993)). Recent reports of NO function in synaptogenesis and in apoptosis during development of the rat CNS (Bredt, D. S., Neuron, 13: 301 (1994); Roskams, A. J., Neuron, 13: 289 (1994)) suggest that NO regulates activity-dependent mechanism(s) underlying the organization of fine-structure in the cortex (Edelman, G. M., et al., Proc. Natl. Acad. Sci. USA, 89: 11651-11652 (1992)). NO also appears to be involved with long-term potentiation in hippocampus and long-term depression in cerebellum, two forms of synaptic plasticity that may underlie behavioral plasticity (Bohme, G. A., Eur. J. Pharmacol., 199: 379 (1991); Schuman, E. M., Science 254: 1503 (1991); O'Dell, T. J., et al., Proc. Natl. Acad. Sci. USA, 88: 11285 (1991); Shibuki, K., Nature, 349: 326 (1991); Haley, J. E., et al., Neuron, 8: 211 (1992); Zhuo, M., Science, 260: 1946 (1993); Zhuo, M., et al., Neuro Report, 5: 1033 (1994)). Consistent with these cellular studies, inhibition of NOS activity has been shown to disrupt learning and memory (Chapman, P. F., et al., Neuro Report, 3: 567 (1992); Holscher, C., Neurosci. Lett., 145: 165 (1992); Bohme, G. A., et al., Proc. Natl. Acad. Sci. USA, 90: 9191 (1993); Rickard, N. S., Behav. Neurosci., 108: 640-644 (1994)).

Many of the above conclusions are based on pharmacological studies using inhibitors of nitric oxide synthases or donors of NO. Interpretations of such studies usually are limited because the drugs interact with more than one target and they cannot be delivered to specific sites. A molecular genetic approach can overcome these problems, however, by disrupting a specific gene, the product of which may be one of the drug's targets. Recently, such an approach has been attempted in mice via generation of a knock-out mutation of the neuronal NOS (nNOS) (Huang, P. L., et al., Cell, 75: 1273-1286 (1993)). While nNOS mutants appeared fully viable and fertile, minor defects in stomach morphology and hippocampal long-term potentiation were detected (Huang, P. L., et al., Cell, 75: 1273-1286 (1993); O'Dell, T. J., et al., Science, 265: 542-546 (1994)). Moreover, some NOS enzymatic activity still was present in certain regions of the brain, suggesting a role for other NOS genes in the CNS. While. yielding some relevant information about one specific component of NO function, this NNOS disruption existed throughout development. Consequently, functional defects of NOS disruption in adults could not be resolved adequately from structural defects arising during development. Genetic tools exist in Drosophila, in contrast, to limit disruptions of gene functions temporally or spatially.

To identify candidate Drosophila NOS homologs, a fragment of the rat neuronal NOS cDNA (Bredt, D. S., et al., Nature, 351: 714-718 (1991)) was hybridized at low stringency to a phage library of the Drosophila genome as described in Example 11. The rat cDNA fragment encoded the binding domains of FAD and NADPH (amino acids 979-1408 of SEQ ID NO.: 11), which are cofactors required for NOS activity, and therefore were expected to be conserved in fruit flies. Several Drosophila genomic clones were identified with the rat probe and classified into eight contigs. Sequence analysis of three restriction fragments from these genomic clones revealed one (2.4R) with high homology to mammalian NOSs. The deduced amino acid sequence of the ORF encoded within the 2.4R fragment indicated 40% identity to the rat neuronal NOS and binding sites for FAD and NADPH.

The 2.4R DNA fragment then was used to probe a Drosophila adult head cDNA library as described in Example 11, and eight clones were isolated. Restriction analysis indicated that all contained identical inserts and thus, defined a predominant transcript expressed by this Drosophila gene. One clone (c5.3) was sequenced in both directions. The 4491 bp cDNA contained one long ORF of 4350 bp. The methionine initiating this ORF was preceded by ACAAG which is a good match to the translation start consensus (A/CAAA/C) for Drosophila genes (Cavener, D. R., Nucleic Acids Res., 15: 1353-1361 (1987)). Conceptual translation of this ORF yielded a protein of 1350 amino acids with a molecular weight of 151,842 Da.

Comparison of the amino acid sequence of this deduced Drosophila protein (DNOS) (SEQ ID NO.: 9) to sequences of mammalian NOSs revealed that DNOS is 43% identical to neuronal NOS (SEQ ID NO.: 11), 40% identical to endothelial NOS (SEQ ID NO.: 10) and 39% identical to macrophage NOS (SEQ ID NO.: 12). It also revealed similar structural motifs in DNOS (FIG. 16A-16C (see Original Patent)). The C-terminal half of the DNOS protein contains regions of high homology corresponding to the presumptive FMN-, FAD- and NADPH-binding sites. Amino acids thought to be important for making contacts with FAD and NADPH in mammalian NOSs (Bredt, D. S., et al., Nature, 351: 714-718 (1991); Lamas, S., et al., Proc. Natl. Acad. Sci. USA, 89: 6348-6352 (1992); Lyons, C. R., et al., J. Biol. Chem., 267: 6370 (1992); Lowenstein, C. J., et al., Proc. Natl. Acad. Sci. USA, 89: 6711 (1992); Sessa, W. C., et al., J. Biol. Chem., 267: 15274 (1992); Geller, D. A., et al., Proc. Natl. Acad. Sci. USA, 90: 3491 (1993); Xie, Q. et al., Science, 256: 225-228 (1992)) are conserved in DNOS. The middle section of DNOS, between residues 215 and 746 of SEQ ID NO.: 9, showed the highest similarity to mammalian NOSs: it is 61% identical to the neuronal isoform and 53% identical to endothelial and macrophage isoforms. Sequences corresponding to the proposed heme- and calmodulin-binding sites in mammalian enzymes are well-conserved in DNOS. The region located between residues 643-671 of SEQ ID NO.: 9 has the characteristics of a calmodulin-binding domain (basic, amphiphilic .alpha.-helix) (O'Neil, K. T., et al., Trends Biochem. Sci., 15: 59-64 (1990)). The amino acid sequence between these two sites is very well conserved among all four NOS proteins, suggesting the location of functionally important domains such as the arginine-binding site (Lamas, S., et al., Proc. Natl. Acad. Sci. USA, 89: 6348-6352 (1992)), tetrahydrobiopterine cofactor binding site or a dimerization domain. DNOS also has a PKA consensus site (Pearson, R. B., Meth. Enzymol., 200: 62-81 (1991)) (at Ser-287 of SEQ ID NO.: 9) in a position similar to neuronal and endothelial NOSs.

The 214 amino acid N-terminal domain of DNOS shows no obvious homology to its equivalent portion of neuronal NOS or to the much shorter N-terminal domains of endothelial and macrophage NOSs. This region of DNOS contains an almost uninterrupted homopolymeric stretch of 24 glutamine residues. Such glutamine-rich domains, found in many Drosophila and vertebrate proteins, have been implicated in protein-protein interactions regulating the activation of transcription (Franks, R. G., Mech. Dev., 45: 269 (1994); Gerber, H.-P., et al., Science, 263: 808 (1994); Regulski, M., et al., EMBO J, 6: 767 (1987)). Thus, this domain of DNOS could be involved with protein-protein interactions necessary for localization and/or regulation of DNOS activity.

The above sequence comparisons suggest that a Drosophila structural homolog of a vertebrate NOS gene was identified. The order of the putative functional domains in the DNOS protein is identical to that of mammalian enzymes (FIG. 15B). Structural predictions based on several protein algorithms also indicate that general aspects of DNOS protein secondary structure (hydrophobicity plot, distribution of .alpha.-helixes and .beta.-strands) from the putative heme-binding domain to the C-terminus are similar to those of mammalian NOSs. DNOS also does not contain a transmembrane domain, as is the case for vertebrate NOSs. In addition to these general characteristics, several aspects of DNOS structure actually render it most like neuronal NOS: (i) the overall sequence similarity, (ii) the similarity of the putative calmodulin-binding site (55% identical to the neuronal NOS vs. 45% identical to endothelial NOS or vs. 27% identical to macrophage NOS) and (iii) the large N-terminal domain. Neuronal NOS and DNOS also do not contain sites for N-terminal myristoylation, which is the case for endothelial NOS (Lamas, S., et al., Proc. Natl. Acad. Sci. USA, 89: 6348-6352 (1992)), nor do they have a deletion in the middle of the protein, which is the case for macrophage NOS (Xie, Q. et al., Science, 256: 225-228 (1992)).

To establish that Applicants putative DNOS protein had nitric oxide synthase activity, the dNOS cDNA was expressed in 293 human embryonic kidney cells as described in Example 12, which have been used routinely in studies of mammalian NOSs (Bredt, D. S., et al., Nature, 351: 714-718 (1991)). Protein extracts prepared from dNOS-transfected 293 cells as described in Example 12, contained a 150 kD polypeptide, which was recognized by a polyclonal antibody raised against the N-terminal domain of DNOS (FIG. 17A, lane 293+dNOS (see Original Patent)). This immunoreactive polypeptide was of a size expected for DNOS and was absent from cells transfected with just the pCGN vector alone (FIG. 17A, lane 293+vector (see Original Patent)).

Extracts made from dNOS-transfected 293 cells showed significant NO synthase activity, as measured by the L-arginine to L-citrulline conversion assay as described in Example 12 (0.1276.+-.0.002 pmol/mg/min; FIG. 17B, group B). [In a parallel experiment, the specific activity of rat neuronal NOS expressed from the same vector in 293 cells was 3.0.+-.0.02 pmol/mg/min, N=4]. DNOS activity was dependent on exogenous Ca.sup.2+/calmodulin and on NADPH, two cofactors necessary for activity of constitutive mammalian NOSs (Iyengar, R., Proc. Natl. Acad. Sci. USA, 84: 6369-6373 (1987); Bredt, D. S., Proc. Natl. Acad. Sci. USA, 87: 682-685 (1990)). DNOS activity was reduced 90% by the Ca.sup.2+ chelator EGTA (FIG. 17B, group C). Also, 500 .mu.M N-(6-aminohexyl)-1-naphthalene-sulfonamide (W5), a calmodulin antagonist which inhibits activity of neuronal NOS (Bredt, D. S., Proc. Natl. Acad. Sci. USA, 87: 682-685 (1990)), diminished DNOS activity to 18% (0.0222.+-.0.001 pmol/mg/min, N=2). In the absence of exogenous NADPH, DNOS (or nNOS) activity was reduced 20% (0.1061.+-.0.011 pmol/mg/min, N=4 for DNOS; 2.7935.+-.0.033 pmol/mg/min, N=2 for NNOS). DNOS activity also was blocked by inhibitors of mammalian NOSs (Rees, D. D., Br. J. Pharmacol., 101: 746-752 (1990)). N.sup.G-nitro-L-arginine methyl ester (L-NAME) reduced DNOS activity 84% (FIG. 17B, group D), and 100 .mu.M N.sup.G-monomethyl-L-arginine acetate produced a complete block (0.0001.+-.0.0002 pmol/mg/min, N=2). These enzymatic data demonstrate that DNOS is a Ca.sup.2+/calmodulin-dependent nitric oxide synthase.

Northern blot analysis indicated a 5.0 kb dNOS transcript which was expressed predominantly in adult fly heads but not bodies (FIG. 18A (see Original Patent)). More sensitive RT-PCR experiments as described in Example 13, however, detected dNOS message in poly(A).sup.+ RNA from fly bodies. Neuronal NOS genes from mice and humans produce two alternatively spliced transcripts, the shorter one of which yields a protein containing a 105 amino acid in-frame deletion (residues 504-608 in mouse or rat neuronal NOS) (Ogura, T., Biochem. Biophys. Res. Commun., 193: 1014-1022 (1993)). RT-PCR amplification of Drosophila head mRNA produced two DNA fragments: the 444 bp fragment corresponded to vertebrate long form and the 129 bp fragment corresponded to vertebrate short form (FIG. 18B). Conceptual translation of the 129 bp sequence confirmed a splicing pattern identical to that for the NNOS gene (FIG. 18C). Presence of the short NOS isoform in Drosophila strengthens the notion that it may play an important role in NOS biochemistry.

The discovery of a NOS homolog in Drosophila provides definitive proof that invertebrates produce NO and, as suggested by recent reports, most likely use it for intercellular signaling. These data also suggest that a NOS gene was present in an ancestor common to vertebrates and arthropods, implying that NOS has existed for at least 600 million years. Thus, it is expected that NOS genes are prevalent throughout the animal kingdom.

Consistent with this view are existing histochemical data. NOS activity has been detected in several invertebrate tissue extracts: in Lymulus polyphemus Radomski, M. W., Philos. Trans. R. Soc. Lond. B. Biol. Sci., 334: 129-133 (1992)), in the locust brain (Elphick, M. R., et al., Brain Res., 619: 344-346 (1993)), in the salivary gland of Rhodniusprolixus (Ribeiro, J. M. C., et al., FEBS Lett., 330: 165-168 (1993)(34)) and in various tissues of Lymnaea stagnalis (Elofsson, R., et al., Neuro Report, 4: 279-282 (1993)). Applications of NOS inhibitors or NO-generating substances have been shown to modulate the activity of buccal motoneurones in Lymnaea stagnalis (Elofsson, R., et al., Neuro Report, 4: 279-282 (1993)) and the oscillatory dynamics of olfactory neurons in procerebral lobe of Limax maximus (Gelperin, A., Nature, 369: 61-63 (1994)). NADPH-diaphorase staining, a relatively specific indicator of NOS protein in fixed vertebrate tissue samples (Dawson, T. M., et al., Proc..sub.--Natl. Acad. Sci. USA, 88: 7797 (1991); Hope, B. T., et al., Proc. Natl. Acad. Sci. USA, 88: 2811 (1991)), also has suggested the presence of NOS in Drosophila heads (Muller, U., Naturwissenschaft, 80: 524-526 (1993)). The present molecular cloning of dNOS considerably strengthens the validity of these observations.

Sophisticated genetic analyses of NOS function are available in Drosophila. Classical genetics will allow the creation of point mutations and deletions in dNOS, resulting in full or partial loss of dNOS function. Such mutations will permit detailed studies of the role of NOS during development.

The invention further relates to isolated DNA that are characterized by their ability to encode a polypeptide of the amino acid sequence in FIG. 16A-16C (SEQ ID NO.: 9 (see Original Patent)) or functional equivalents thereof (i.e., a polypeptide which synthesizes nitric oxide). Isolated DNA meeting this criteria comprise amino acids having sequences homologous to sequences of mammalian NOS gene products (i.e., neuronal, endothelial and macrophage NOSs). The DNA sequence represented in SEQ ID NO.: 25 is an example of such an isolated DNA. Isolated DNA meeting these criteria also comprise amino acids having sequences identical to sequences of naturally occurring dNOS or portions thereof, or variants of the naturally occurring sequences. Such variants include mutants differing by the addition, deletion or substitution of one or more residues, modified nucleic acids in which one or more residues is modified (e.g., DNA or RNA analogs), and mutants comprising one or more modified residues.

Such nucleic acids can be detected and isolated under high stringency conditions or moderate stringency conditions, for example. "High stringency conditions" and "moderate stringency conditions" for nucleic acid hybridizations are explained on pages 2.10.1-2.10.16 (see particularly 2.10.8-11) and pages 6.3.1-6 in Current Protocols in Molecular Biology (Ausubel, F. M. et al., eds, Vol. 1, Suppl. 26, 1991), the teachings of which are incorporated herein by reference. Factors such as probe length, base composition, percent mismatch between the hybridizing sequences, temperature and ionic strength influence the stability of nucleic acid hybrids. Thus, high or moderate stringency conditions can be determined empirically, depending in part upon the characteristics of the known DNA to which other unknown nucleic acids are being compared for homology.

Isolated DNA that are characterized by their ability to encode a polypeptide of the amino acid sequence in FIG. 16A-16C (see Original Patent), encode a protein or polypeptide having at least one function of a Drosophila NOS, such as a catalytic activity (e.g., synthesis of nitric oxide) and/or binding function (e.g., putative heme, calmodulin, FMN, FAD and NADPH binding). The catalytic or binding function of a protein or polypeptide encoded by hybridizing nucleic acid may be detected by standard enzymatic assays for activity or binding (e.g., assays which monitor conversion of L-arginine to L-citrulline). Functions characteristic of dNOS may also be assessed by in vivo complementation activity or other suitable methods. Enzymatic assays, complementation tests, or other suitable methods can also be used in procedures for the identification and/or isolation of nucleic acids which encode a polypeptide having the amino acid sequence in FIG. 16A-16C or functional equivalents thereof.
 

Claim 1 of 50 Claims

1. A method for assessing the effect of a drug on long term memory formation comprising: a) administering said drug to an animal having an inducible activator and/or an inducible repressor wherein said inducible activator and/or inducible repressor is/are under the control of a promoter, wherein said activator is a CREB/CREM/ATF-1 subfamily member associated with potentiation of long term memory and said repressor is an antagonist of said activator and is associated with blocking of long term memory; b) inducing expression of said activator and/or repressor, wherein inducing expression of said activator and/or repressor comprises activating said promoter effective to induce expression of said activator and/or repressor; c) training said animal under conditions appropriate to produce long term memory formation in said animal, wherein conditions appropriate to produce long term memory formation comprise multiple training sessions with a rest interval between training sessions; d) assessing long term memory formation in said animal trained in step c); and e) comparing long term memory formation assessed in step d) with long term memory formation produced in a control animal to which said drug has not been administered.

 

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