Methods have been developed for alleviating memory problems or psychiatric dysfunctions that have a memory formation component. These methods are based on the finding that a truncated form of an aPKC.zeta. protein is intimately involved in memory formation in animals. This finding is also central to methods for determining drugs that will have an effect on memory formation or the memory formation component of psychiatric dysfunctions.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention is directed to methods of alleviating memory problems and psychiatric dysfunctions in mammals by modulating the expression, or activity, of a truncated form of an aPKC ZETA (aPKC.zeta.) protein in the central nervous system of the mammal. A particular trucated form of the aPKC.zeta. protein that is responsive to modulation is aPKM.zeta.. Modulation of protein expression can be achieved by either induction or inhibition of formation of aPKM.zeta.. Modulation of activity can be achieved by affecting interacting proteins that participate in the normal memory formation process. Memory problems include abnormal memory formation due to normal aging, injury to the brain, neurodegeneration, and Alzheimer's disease or other decreases in cognitive ability. The present invention alleviates memory deficits associated with these situations. In this instance, the problem is alleviated by inducing expression of aPKM.zeta. and memory formation is particularly improved when the expression of aPKM.zeta. is induced. Psychiatric dysfunctions for which alleviation can be achieved include attention deficit disorder, autism, fragile X syndrome, bipolar disorder, schizophrenia, obsessive compulsive disorders and phobias. In this regard, the psychiatric dysfunctions are alleviated by affecting or altering the memory formation component of the particular dysfunction.

The present invention is also directed to methods of identifying substances that can affect memory formation or psychiatric dysfunctions in mammals. In these methods, a substance under scrutiny is administered to a mammal. It is then determined whether the substance alters the expression or activity of aPKM.zeta. protein in the central nervous system of the mammal when compared to the expression or activity of the same protein in the absence of the substance. If there is a difference in such expression or activity, and the aPKM.zeta. protein is associated with a memory defect or a given psychiatric dysfunction, the substance affects that defect or dysfunction. The portion of the psychiatric dysfunction that is affected when the substance alters the expression or activity of the aPKM.zeta. protein is the memory formation component of the dysfunction. In particular instances, an increase in expression or activity of the aPKM.zeta. protein when the substance is administered is indicative that the substance will enhance the memory formation component of dysfunction. By contrast, a decrease in expression or activity of the aPKM.zeta. protein when the substance is administered is indicative that the substance will interfere with the memory formation component of the dysfunction.

The present invention is further directed to methods for assessing the effect of a drug on regular memory disorders or the memory formation component of a psychiatric dysfunction. To test the effect of a drug on regular memory disorders, animal models for a memory disorder are trained and tested in the presence and absence of the drug. If the drug affects performance relative to the performance of animals that were trained identically but in a drug-free state, then the drug has an effect on regular memory disorders. Regular memory disorders result from the processes of normal aging, traumatic injury to the brain, Alzheimer's disease, and neurodegeneration. To test the effect of the drug on the memory formation component of a psychiatric dysfunction, the drug is administered to an animal that has an animal model for a specified psychiatric dysfunction. The animal is subjected to a training protocol and the performance index of the animal is assessed. The drug is found to have an effect on memory formation when the performance significantly differs between drug-free and drug-treated animals that were trained identically. Psychiatric dysfunctions which have a memory formation component and for which the effects of drugs can be assessed by these methods include attention deficit disorder, autism, Fragile X syndrome, bipolar disorder, schizophrenia, obsessive compulsive disorder and phobias.

The present invention is further directed to methods of alleviating regular memory disorders and psychiatric dysfunctions in mammals by modulating the expression of an aPKM.zeta. gene that is associated with the regular memory disorder or the psychiatric dysfunction. When the modulation is induction of the aPKM.zeta. gene, the induction enhances the normal memory formation process, or the memory formation component of a psychiatric dysfunction. Regular memory disorders result from normal aging, traumatic injury to the brain, Alzheimer's disease and neurodegeneration. Psychiatric dysfunctions for which alleviation can be achieved by modulation of an aPKM.zeta. gene include attention deficit disorder, autism, Fragile X syndrome, bipolar disorder, schizophrenia, obsessive compulsive disorder, and phobias.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to methods of alleviating memory problems and psychiatric dysfunctions in mammals, particularly in humans, by modulating the expression or activity of a truncated form of an aPKC.zeta. protein in the central nervous system of the mammal. The expression of the truncated form of the aPKC.zeta. protein is modulated by affecting molecular processes of transcription, mRNA stability, protein stability, proteolytic processing, and translation initiation. The activity of the protein is modulated by post-translational modifications on the protein, protein:protein interactions, and subcellular localization of the protein.

Modulation of expression occurs when the amount of the truncated form of the aPKC.zeta. protein differs from the amount that is present without modulation. There can be an increase in the amount of truncated form of the aPKC.zeta. protein, decrease in the amount of truncated form of the aPKC.zeta. protein, or a variation over a defined time span of an increase and/or a decrease in the amount of truncated form of the aPKC.zeta. protein. For example, the amount of truncated form of the aPKC.zeta. protein can increase in a linear or nonlinear fashion over time, decrease in a linear or nonlinear fashion, or alternatively increase and decrease, linearly or nonlinearly. An increase in the amount of the truncated form of the aPKC.zeta. protein, or in the amount of an inhibiting form of the protein, in a linear or nonlinear fashion, is generally preferred for the alleviation of psychiatric dysfunctions.

The protein that is formed by the expression process in this invention is a truncated form of an aPKC.zeta. protein. This protein is produced either by synthesis of the full-length protein, followed by proteolytic processing, or de novo synthesis of the truncated form of the protein, aPKM.zeta.. This de novo synthesis may occur from transcription initiation sites located in intronic regions of the gene, and/or translation initiation from internal methionine codons.

Modulation of the expression of the truncated form of an aPKC.zeta. protein can occur either by an induction or by an inhibition of the expression of the truncated form. When induction of expression occurs, more of the truncated form is produced than when this induction is not present. When inhibition of expression occurs, less of the truncated form is produced than when this inhibition is not present. Under certain conditions, a given amount of the truncated form of the aPKC.zeta. protein normally may be produced. Under these conditions, induction of expression increases the amount of the truncated form that is produced and inhibition of expression decreases the amount of the truncated form that is produced from this given amount. In these instances, modulation of the expression of the truncated form causes an increase, decrease, or alternative increase and decrease, linearly or nonlinearly, from the given amount that is normally present.

In this invention, the preferred truncated form of the aPKC.zeta. protein is aPKM.zeta.. This is the truncated form of the a typical isozyme PKC.zeta. that lacks the N-terminal regulatory domain of the PKC.zeta. protein. The N-terminal regulatory domain contains a pseudosubstrate region as well as binding sites for the required cofactors. The aPKM.zeta., which lacks this N-terminal regulatory domain, contains the C-terminal catalytic domain and is a persistently active kinase derived from the aPKC.zeta. isozyme. Induction of the expression of aPKM.zeta. protein or inhibition of the expression of the aPKM.zeta. protein are preferred in this invention to alleviate psychiatric disorders. Of these, induction of expression is most preferred.

Normal memory problems can be alleviated by modulating the expression or activity of aPKM.zeta.. These memory problems can result from normal aging, traumatic injury to the brain, Alzheimer's disease and neurodegeneration. Classical memory disorders include loss or lack of ability to recall specific past experiences or events. The loss or lack of ability to make proper or normal associations between these prior events or past experiences is also included in these disorders. These disorders also include the loss or lack of ability to make proper or normal associations between prior events or past experiences and present cognitive functions or experiences. Short term memory loss and long term memory loss are particular memory deficits that are included in these disorders. Short term memory losses are the loss or lack of ability to recall or make correct or proper associations between present perceptions and recent events or experiences. Long term memory losses are the loss or lack of ability to recall or make correct or proper associations between present perceptions and events or experiences that were perceived some time ago by the individual. The distinction between short term memory and long term memory varies with the animal species, behavioral task, and training regimen, and is generally known to people to whom this distinction is important. For all animals, long-term memory is the memory phase whose induction is sensitive to protein synthesis inhibitors given acutely around the time of training. Short-term memory are all phases of memory that are resistant to such inhibitors. Generally, short term memories last from minutes, to hours and a few days after training, while long-term memories persist for longer periods of time.

Many memory problems and psychiatric dysfunctions can be alleviated by modulating the expression of aPKM.zeta. in animals. Alleviation of a given psychiatric dysfunction occurs when the symptoms of the dysfunction are lessened and the individual exhibits more normal behavior patterns and modes. In certain instances, and usually desired, alleviation of a given psychiatric dysfunction is essentially complete and the individual exhibits normal behavior. In rare instances, the individual exhibits normal behavior traits before the expression of aPKM.zeta. protein is modulated. Under these circumstances, better than normal behavior is sought and expression of the truncated form is modulated to achieve this result.

Among the memory problems that can be alleviated by modulating the expression of aPKM.zeta. are those resulting from normal aging, injury to the brain, Alzheimer's disease or neurodegeneration. Among the psychiatric dysfunctions that can be alleviated by modulating the expression of aPKM.zeta. are attention deficit disorder, autism, fragile X syndrome, bipolar disorder, schizophrenia, obsessive compulsive disorder, and phobias.

In the present invention, memory is affected by modulation of expression of the aPKM.zeta. protein. Depending upon the protein isoform that is modulated, memories can be enhanced or blocked. The aPKM.zeta. appears to have a noticeable effect on short term memory when its expression is modulated. Short term memory improves when expression of aPKM.zeta. is induced.

Without being bound by any mechanism of action, it appears that the psychiatric dysfunctions that are alleviated by modulating the expression of a truncated form of aPKM.zeta. in the central nervous system of the animal are aided or relieved because the modulation affects the memory formation component of the psychiatric dysfunction. By making the appropriate changes to the memory formation component, the symptoms of the psychiatric dysfunction are lessened and the psychiatric dysfunction is altered in a favorable manner. In many instances, it is the short term memory component of the psychiatric dysfunctions that is affected by modulating the expression of the APKM.zeta. protein. Induction of expression of the aPKM.zeta. causes an enhancement of the short term memory component of the psychiatric dysfunction. Inhibition of expression or activity of aPKM.zeta. causes an interference with the short term memory component of the psychiatric dysfunction. Either an enhancement or interference with the short term memory component can alleviate a given psychiatric dysfunction. Whichever process is desired to alleviate the psychiatric dysfunction will be employed. For example, when an individual exhibits a lack or loss of short term memory ability, the expression of aPKMC can be induced to relieve symptomology.

The amount of truncated form of aPKC.zeta. protein in the central nervous system of the animal can be changed or modulated in a variety of manners. Transcription of the endogenous gene can be modulated. Any small molecule or physiological stimulus that affects the amounts or activity of the different transcription factors that modulate gene expression will affect levels of the mRNA and protein. Alternatively, DNA based manipulations can be performed to change the regulation of the endogenous gene. Exogenous regulatory sequences can be added to the endogenous gene, putting the gene under the control of different DNA sequences, proteins that bind those sequences, and effectors that affect those proteins. In these situations, modulation of expression of the truncated form occurs when the effector is administered from an external source or withheld, depending on the action that occurs at the regulation site.

Another manner of changing or modulating the amount of aPKM.zeta. protein in the central nervous system of the animal is using transgenic technology. A transgene that encodes a desired aPKM.zeta. protein is inserted into the genome of the animal. The transgene can be inserted using recombinant techniques recognized and known to skilled persons such as molecular biologists. The transgenic animal can contain one or more copies of the transgene that encodes the truncated form of the aPKM.zeta. protein. This transgene may contain the endogenous gene. More likely, the transgene encodes a selected aPKM.zeta. protein of another animal species. In either instance, the transgene can be under the control of either endogenous regulation sites or regulation sites obtained from exogenous sources. Endogenous regulation sites can be employed when the transgene is inserted at an appropriate locus in the genome where gene expression is controlled by the endogenous regulation site. However, regulation sites from exogenous sources are more often employed when transgenes are used. The regulation sites are often easier to include with the transgenes when the genome insertions are performed. In either situation, the inserted transgene encoding the desired aPKM.zeta. provides more control of the modulation, particularly induction, of the expression of the truncated form. This increased control enhances the ability to alleviate memory defects and psychiatric dysfunctions.

A further manner of changing the amount of aPKM.zeta. protein in an individual is by administration of the protein itself to the individual. The protein is administered so that it is active in the central nervous system of the individual and thereby alleviates the memory defect or psychiatric dysfunction by altering the memory formation component of the dysfunction. This protein can be an active or inhibitory form of the molecule.

This invention also relates to methods of identifying substances that affect memory formation or psychiatric dysfunctions in mammals, particularly in humans. The substance usually has an organic chemical structure and is in the form of a pharmaceutical with the required diluents, excipients and carriers present in its formulation. The substance may be a macromolecule but usually it is much smaller. In these methods, the substance under consideration is administered to a mammal. The administration is by any standard route. For example, administration can occur by oral or rectal intake, inhalation, topical application, or parenterally by subcutaneous, intravenous or intramuscular injection. Once administered, it is determined whether the substance alters the expression or activity of an aPKM.zeta. protein in the central nervous system of the mammal, where it has previously been shown that the aPKM.zeta. protein is associated with the psychiatric dysfunction of interest. The association of the aPKM.zeta. protein and the psychiatric dysfunction can be direct or indirect. The association is present if an alteration in the amount or activity of the aPKM.zeta. protein either enhances or diminishes the signs or symptoms of the psychiatric dysfunction. The association is present if an alteration in the genetic expression of the aPKM.zeta. protein either enhances or diminishes the signs or symptoms of the psychiatric dysfunction. Alteration of expression or activity of the aPKM.zeta. protein is determined by comparing the expression or activity of this protein after the substance is administered to the mammal with the expression of the protein when the substance has not been administered. If a reproducible difference is found between the expression or activity values for the aPKM.zeta. protein when the substance is present versus when the substance is not present, the substance is identified as having the property of affecting the psychiatric dysfunction with which the aPKM.zeta. protein is associated. The substance can be further identified as having an alleviating or a deleterious effect on the psychiatric dysfunction, depending on the relationship of the expression change with the quality or intensity of the signs or symptoms of the psychiatric dysfunction. For example, if an increase in the expression or activity of the aPKM.zeta. protein is associated with an alleviation of the signs or symptoms of the psychiatric dysfunction and the substance, when administered, causes an increase in the expression or activity of the aPKM.zeta. protein, the substance is considered to have advantageous properties for alleviating the psychiatric dysfunction.

Often for aPKM.zeta., when the administered substance causes an increase in the expression or activity of the APKM.zeta., the substance is considered to have the property of enhancing the memory formation component of the psychiatric dysfunction. Often, it is the short term memory component of the psychiatric dysfunction that is enhanced when the administered substance causes an increase in the expression of aPKM.zeta.. Conversely, when the administered substance causes a decrease in the expression or activity of the aPKM.zeta., the substance is considered to have the property of interfering with the memory formation component of the psychiatric dysfunction. In this instance, it is again often the short term memory formation component of the psychiatric dysfunction that is interfered with or blocked by the administered substance.

This invention further relates to methods for assessing the effects of drugs on the memory formation component of psychiatric dysfunction. In these methods, the candidate drug is administered to a normal animal or an animal that possesses an inducible aPKM.zeta. protein which is associated with memory formation. The drug usually has an organic chemical structure and is administered by any of the standard administration routes together with any required diluents, excipients or carriers. In these methods, the animal type is not limited to mammals but includes most of the animal kingdom. For example, insects such as Drosophila melanogaster or honeybees can be used as subjects for assessing the effects of drugs on the memory formation component. Other model organisms for assessing drug effects on memory formation include C. elegans, Aplysia, Xenopus, zebrafish, mouse, rats, ferrets and cats. The only requirement for animal type is that it have an inducible truncated form of an aPKC.zeta. protein which is associated with memory formation.

Following the administration of the candidate drug to the animal in these methods, the inducible aPKM.zeta. protein is induced to produce the truncated form in the animal, or the endogenous gene is examined in the nontransgenic animal. Induction can be performed by any of the methods known to persons who are familiar with such processes. In C. elegans, practitioners usually use heat-shock, antibiotics or small molecules such as IPTG. In Drosophila, practitioners usually use heat-shock, antibiotics, or small molecules like IPTG or heavy metals. In mammals, practitioners usually use antibiotics, hormones or small molecules like IPTG.

After the expression of the aPKM.zeta. protein has been induced in the transgenic animal, or the endogenous gene in the normal animal, the animal is subjected to a learning and memory assay, and a performance index, based on the outcome of the protocol, is assigned. Learning and memory tests are known to psychologists and others who study learning and memory in animals. The training protocols that are useful in this invention address learning and memory attributes which the animal possesses. Any discriminative classical conditioning protocol can be used. Exemplary of these protocols are associative and non-associative conditioning protocols, classical and operant conditioning, and tests of implicit and explicit memory. The performance index is an assessment of the results of the training protocol that was used. Often the performance index is a numerical value that is assigned by the observer or investigator to the outcome of the training protocol. The numerical value can be considered to be a scaled score for the performance of the animal undergoing the classical conditioning protocol.

In these methods, the drug is considered to have an effect on memory formation or the memory formation component of the psychiatric dysfunction when animals treated with the compound reproducibly perform differently from untreated animals. In some instances, the same individual animal may serve as the control animal and the one to whom the drug is administered. Usually, however, different animals serve as the untreated (control) and treated (subject) animals. In these instances, the animals should be chosen from the same cohort. The drugs that exhibit an effect on the memory formation component, as detected by these methods, are candidates for administration to animals to alleviate psychiatric dysfunctions in these animals, particularly when the psychiatric dysfunction has a memory formation component.

In these methods, the inducible target of the drug is aPKM.zeta.. In these instances, it is typically the short term memory formation component of the memory problem or psychiatric dysfunction that is affected by the drug. Memory problems targeted by the selected drugs include those resulting from normal aging, injury to the brain, Alzheimer's disease and neurodegeneration. Psychiatric dysfunctions for which the drugs selected by these methods will have an effect include attention deficit disorder, autism, fragile X syndrome, bipolar disorder, schizophrenia, obsessive compulsive disorders, and phobias.

The study of PKC in memory formation has a long history. However, most previous studies were done before the complexity of the PKC gene family was appreciated. The PKC family can be divided into three classes based on their cofactor requirements. Whereas all PKC proteins require phosphatidylserine for activation, the `conventional` (cPKC) isotypes require diacylglycerol (DAG) and Ca2+ for full activity; `novel` (nPKC) isotypes are Ca2+ independent but still require DAG, and the `atypical` (aPKC) isotypes are both DAG and Ca2+ independent. Structurally, these kinases can be divided into an N-terminal regulatory domain, which contains a pseudosubstrate region as well as the binding sites for the required cofactors, and the C-terminal catalytic domain. Removal of the N-terminal regulatory domain produces a persistently active kinase, referred to as PKM.

The roles of PKC in hippocampal models of synaptic plasticity, long-term potentiation (LTP), and long-term depression (LTD) have been studied extensively (see, F. Angenstein, et al., Prog. Neuropsychopharmacol. Biol. Psychiatry 21, 427-454 (1997)). Western blot analyses with antibodies specific for each of the rat PKC isoforms demonstrate that the only one whose levels specifically increase and remain elevated during the maintenance phase of LTP is PKM.zeta., the truncated form of the a typical isozyme PKC.zeta.(see, e.g., Osten, P. et al., J Neurosci. 16, 2404-2451 (1996)). Expression analyses also show that the maintenance of LTD is associated with decreasing levels of PKM.zeta.. Most interestingly, LTP maintenance is abolished by sustained application of low concentrations of the PKC inhibitor chelerythrine, whereas perfusion of PKM.zeta. into CA1 pyramidal cells produces an increase in AMPA receptor-mediated synaptic transmission (D. S. F. Ling et al, unpublished data).

In Drosophila, the best characterized assay for associative learning and memory is an odor-avoidance behavioral task (T. Tully, et al. J. Comp. Physiol. A157, 263-277 (1985) incorporated herein by reference). This classical (Pavlovian) conditioning involves exposing the flies to two odors (the conditioned stimuli, or CS), one at a time, in succession. During one of these odor exposures (the CS+), the flies are simultaneously subjected to electric shock (the unconditioned stimulus, or US), whereas exposure to the other odor (the CS-) lacks this negative reinforcement. Following training, the flies are then placed at a `choice point`, where the odors come from opposite directions, and expected to decide which odor to avoid. By convention, learning is defined as the fly's performance when testing occurs immediately after training. A single training trial produces strong learning: a typical response is that >90% of the flies avoid the CS+. Performance of wild-type flies from this single-cycle training decays over a roughly 24-hour period until flies once again distribute evenly between the two odors. Flies can also form long-lasting associative olfactory memories, but normally this requires repetitive training regimens.

This task in Drosophila was used to examine in an exemplary fashion herein the role of atypical PKM in memory formation. Induction of the mouse aPKM.zeta. (MaPKM.zeta.) transgene enhances memory, and corrects the memory defect of radish mutants. There is a single atypical PKC in Drosophila, and the truncated `M` isoform, DaPKM, was found to be preferentially expressed and active in fly heads. Both pharmacological and dominant-negative genetic intervention of DaPKC/M activity disrupt normal memory. Finally, induction of the predicted DaPKM also enhances memory, further demonstrating a general role of aPKM in memory processes.
 

Claim 1 of 14 Claims

1. A method of identifying a substance that affects a short term memory problem or a short term memory formation component of a psychiatric dysfunction associated with atypical PKM zeta (aPKM.zeta.) protein in a mammal, comprising: (a) administering said substance to a mammalian animal model; and (b) determining whether said substance alters the expression or activity of an aPKM.zeta. protein in the central nervous system of said mammalian animal model when compared to the expression or activity of said aPKM.zeta. protein in the absence of said substance; wherein a change in the expression or activity of said aPKM.zeta. in the presence of said substance, compared to the expression or activity of said aPKM.zeta. in the absence of said substance, is indicative that said substance affects short term memory formation or said short term memory formation component of said psychiatric dysfunction.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.