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Link:  Pharm/Biotech Resources


Title:  Method for assessing the effect of a drug on long term memory formation

United States Patent:  6,890,516

Issued:  May 10, 2005

Inventors:  Tully; Timothy P. (Cold Spring Harbor, NY); Yin; Jerry Chi-Ping (Huntington, NY)

Assignee:  Cold Spring Harbor Laboratory (Cold Spring Harbor, NY)

Appl. No.:  419371

Filed:  October 14, 1999

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

BACKGROUND OF THE INVENTION

Activation of the cyclic 3′,5′-adenosine monophosphate (cAMP) signal transduction pathway can have long-lasting global consequences through its influence on the expression of specific genes. This is true for simple organisms as well as mammals, where many of the known cAMP-responsive genes can have important neural and endocrine roles. Additional information regarding activation of this pathway would be useful, particularly as this activation pertains to the ability of animals to remember activities or events.

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 (ΔC) 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 (ΔC) 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 (ΔC) 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 (ΔC). 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 (ΔC). 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).

Claim 1 of 24 Claims

1. A method for assessing the effect of a drug on long term memory formation comprising:

a) administering said drug to an animal; and

b) determining the functional level of activator and repressor in said animal relative to the functional level of activator and repressor in a control animal to which said drug has not been administered, wherein said activator is a CREB/CREM/ATF-1 subfamily member associated with potentiation of long term memory and said repressor is a CREB/CREM/ATF-1 subfamily member associated with blocking of long term memory.

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