5 FIELD OF INVENTION

[0001] This invention relates to methods for improving cognitive function in old age. The methods are characterized by compounds that particularly attenuate the function or reduce the abundance of a voltage-gated potassium channel. Compounds having that activity are known or can be identified using the assays of 10 this invention.

BACKGROUND OF THE INVENTION

[0002] Cognitive and/or degenerative brain disorders are characterized clinically by progressive loss of memory, cognition, reasoning, judgment and emotional

15 stability that gradually leads to profound mental deterioration and ultimately death. Among these diseases, Alzheimer's disease is considered as the most common and is believed to represent the fourth most common medical cause of death in the United States. In 1997, Alzheimer's disease was estimated to affect more than 2 million people in the United States, a number expected to quadruple within the

20 next 50 years (Brookmeyer et al., 1998). Even more patients, approximately three times as many as Alzheimer's disease patients, are estimated to suffer from Mild Cognitive Impairment (Yesavage et al., 2002; Hanninen et al., 2002). Additionally, the number of patients falling in the category of Age- Associated Memory Impairment or similar diagnostic categories (e.g., Age-Related Cognitive 5 Decline) is staggering. For example, according to the estimates of Barker et al. (1995) there are more than 16 million people with Age Associated Memory Impairment in the U.S. alone.

[0003] Extensive behavioral characterization has identified a naturally occurring form of cognitive impairment in an outbred strain of aged Long-Evans rats. An important feature of this model is that it mirrors the phenomenon of variability in cognitive decline among elderly humans. Furthermore, the individual differences in cognitive decline in aged rats in this model are seen in a behavioral assessment that is sensitive to the function of interconnected structures in the medial temporal lobe, a system that is essential for declarative memory in humans. In the behavioral assessment, rats learn and remember the location of an escape platform guided by a configuration of spatial cues surrounding the maze. Aged rats in the study population have no difficulty swimming to a visible platform, but an age- dependent impairment occurs when the platform is camouflaged, requiring the use of spatial information. As reported in earlier publications (Sung et al. 2008 or Bizon et al. 2004, for example), performance for individual aged rats varies greatly, with a proportion of those rats performing on a par with young adults but approximately 40-50% falling outside the range of young performance. This variability among aged rats reflects reliable individual differences. In a reassessment in a new spatial environment several weeks after the original characterization, an aged subgroup is consistently impaired, whereas the aged rats that had preserved behavioral function in the first assessment again perform proficiently. This naturally occurring variability of individual differences in an aged population of rodents indicates that cognitive aging is not inevitable or strictly linked to chronological age, and, importantly, it affords the opportunity to compare the trajectory of changes in the brain that lead to a decline or preservation of memory processes.

SUMMARY OF THE INVENTION

[0004] In accordance with a first aspect of the present invention, there is provided a method for identifying a compound useful in the treatment of cognitive impairment comprising the steps of measuring the function or abundance of a voltage-gated potassium channel or a protein subunit thereof in a eukaryotic cell that expresses the voltage-gated potassium channel or the protein subunit thereof in the presence of a candidate compound, measuring the function or abundance of the voltage-gated potassium channel or the protein subunit thereof in the eukaryotic cell in the absence of said candidate compound, and identifying a compound from among the candidate compound(s) that attenuates the function, or reduces the abundance, of the voltage-gated potassium channel or the protein subunit thereof.

[0005] In certain embodiments of the invention, the eukaryotic cell expresses the voltage-gated potassium channel or the protein subunit thereof in a functional form.

[0006] In accordance with a second aspect of the present invention, there is provided a method for identifying a compound useful in the treatment of cognitive impairment comprising the steps of determining the cognitive status of a mammal with cognitive impairment treated with a candidate compound believed to attenuate the function or to reduce the abundance of a voltage-gated potassium channel or a protein subunit thereof, determining the cognitive status of the mammal with cognitive impairment in the absence of treatment with said candidate compound, and identifying a compound from among the candidate compound(s) that beneficially alters the cognitive status of the mammal with cognitive impairment. [0007] In certain embodiments of the invention, the cognitive status analyzed in the method is a hippocampus-dependent function. In certain embodiments of the invention, the hippocampus-dependent function is selected from the group consisting of spatial memory acquisition, long-term spatial memory, and spatial memory retrieval. [0008] In certain embodiments of the invention, the compound beneficially alters the cognitive status of an aged cognitively impaired mammal and does not significantly alter the cognitive status of a member of the group consisting of a young mammal, an aged cognitively unimpaired mammal, and both.

[0009] In certain embodiments of the methods of identifying compounds of the invention, the mammal is a rat. [0010] In accordance with a third aspect of the present invention, there is provided a method for treating cognitive impairment in a human subject in need thereof, comprising the step of administering to said subject a pharmaceutical composition comprising a therapeutically effective amount of a compound that attenuates the function or reduces the abundance of a voltage-gated potassium channel or a protein subunit thereof.

[0011] In certain embodiments of the methods of treating cognitive impairment of this invention, the cognitive impairment is selected from the group consisting of Mild Cognitive Impairment (MCI), Age-Related cognitive Decline (ARCD) and Age- Associated Memory Impairment (AAMI).

[0012] In certain embodiments of the methods of identifying compounds of and treating cognitive impairment of this invention, the voltage-gated potassium channel is selected from the group consisting of an A-type potassium channel, a Shal potassium channel, a Kv4 potassium channel, a Kv4.1 potassium channel, a Kv4.2 potassium channel and a Kv4.3 potassium channel. In certain embodiments of the methods of identifying compounds of and treating cognitive impairment of this invention, the protein subunit of a voltage-gated potassium channel is selected from the group consisting of Kv4.2, DPP6 and Kcnip2.

[0013] In certain embodiments of the identification methods and treatment methods of this invention, the compound alters the functional interaction of potassium ion channel proteins. In certain embodiments of the invention, the compound alters the functional interaction of proteins selected from the group consisting of Kv4.2, DPP6 and Kcnip2.

[0014] In certain embodiments of the treatment methods of this invention, the compounds are identified in the identification methods of the invention. In other embodiments of the treatment methods of this invention, the compounds are selected from the group consisting of Heteropodatoxin- 1 , Heteropodatoxin-2, Heteropodatoxin-3 and alpha-KTxl5.

DESCRIPTION OF THE FIGURES [0015] Figure 1. Affymetrix GeneChip Rat Expression Data. Data were obtained using Affymetrix GeneChip Rat Expression Set 230 for the dissected CA3 region of the hippocampus. After extensive microarray quality assessment, data were analyzed for 8 young, 8 aged impaired and 7 aged unimpaired rats. Multidimensional scaling eliminated two additional outlier AU chips to yield N=5 for aged unimpaired. Probe set intensities were normalized using GCRMA and statistical analysis performed on these data using the Significance Analysis in Microarrays (SAM).

[0016] Figure 2. Kv4.2 gene expression compared to learning index for aged impaired and aged unimpaired animals. A. Logarithmic comparison of Kv4.2 gene expression levels in the dorsal hippocampus among young, aged unimpaired rats and aged impaired rats. Kv4.2 gene expression level of aged unimpaired (AU) rats is significantly lower than that of young (Y) and aged impaired (AI) rats.

B. Linear regression analysis of the relationship between Kv4.2 mRNA levels and the learning index between aged-impaired animals (♦ filled diamond) and aged- unimpaired animals (0 empty diamond). Levels of Kv4.2 gene expression in the hippocampus of aged Long-Evans rats plotted versus the learning index. The x- axis is the scale of the learning indices, with better performance in the spatial task at the left end and poorer performance at the right end. Lower scores on the index indicate a more accurate search in the vicinity of the target location; higher scores indicate a more random search and poor learning.

[0017] Figure 3. Kcnip2 gene expression compared to learning index for aged impaired and aged unimpaired animals. A. Comparison of Kcnip2 gene expression levels in the dorsal hippocampus among young (Y), aged unimpaired (AU) rats and aged impaired (Al) rats. Kcnip2 gene expression level of AU rats is significantly lower than that of young or aged impaired rats.

B. Logarithmic linear regression analysis of relationships between Kcnip2 mRNA levels and the learning index between aged-impaired animals (♦ filled diamond) and aged-unimpaired animals (0 empty diamond). Levels of Kcnip2 gene expression in the dorsal hippocampus of aged Long-Evans rats, plotted versus the learning index. The ;c-axis is the scale of the learning indices, with better performance in the spatial task at the corresponding to lower values and poorer performance at the corresponding to higher values. Lower scores on the index indicate a more accurate search in the vicinity of the target location; higher scores indicate a more random search and poor learning. [0018] Figure 4. DPP6 gene expression compared to learning index for aged impaired and aged unimpaired animals. A. Comparison of DPP6 gene expression levels in the dorsal hippocampus among young (Y), aged unimpaired (AU) rats and aged impaired (AI) rats. DPP6 gene expression level of AU rats is significantly lower than that of young or aged impaired rats. B. Logarithmic linear regression analysis of relationships between DPP6 mRNA levels and the learning index. Levels of gene expression of DPP6 in the dorsal hippocampus of aged Long-Evans rats, plotted versus the learning index between aged-impaired animals (♦ filled diamond) and aged-unimpaired animals (0 empty diamond). The x-axis is the scale of the learning indices, with better performance in the spatial task at the corresponding to lower values and poorer performance at the corresponding to higher values. Lower scores on the index indicate a more accurate search in the vicinity of the target location; higher scores indicate a more random search and poor learning.

[0019] Figure 5. Kv4.2 protein expression compared to learning index for aged impaired and aged unimpaired animals. A. Comparison of Kv4.2 protein expression levels in the dorsal hippocampus among young, aged unimpaired rats and aged impaired rats. Kv4.2 protein expression level of aged unimpaired (AU) rats is significantly lower than that of young (Y) and aged impaired (AI) rats.

B. Linear regression analysis of the relationship between Kv4.2 protein levels and the learning index between aged-impaired animals (A filled triangles) and aged- unimpaired animals (■ filled square). Levels of Kv4.2 protein expression in the hippocampus of aged Long-Evans rats plotted versus the learning index. The x- axis is the scale of the learning indices, with better performance in the spatial task at the left end and poorer performance at the right end. Lower scores on the index indicate a more accurate search in the vicinity of the target location; higher scores indicate a more random search and poor learning. C. Kv4.2 protein expression in CA3b plotted against distance of dendrite from soma. Error bars indicate SEM.

DETAILED DESCRIPTION OF THE INVENTION [0020] Changes in gene expression in essential memory circuits that occur in aged rats with high performing cognitive function are potential entry points for therapies to treat agc-rclated cognitive decline. We have disclosed changes in mRNA in aged unimpaired rats on an individual gene basis (See Int'l Pub. No. WO 2007/019312, for example). Determination of a coordinated change in a cluster of mRNAs expressing proteins that form a functional unit are additional entry points for intervention to modify multiple components of a functional unit or interactions among components that control function of the unit.

[0021] Gene expression arrays offer the potential to simultaneously analyze thousands expressed genes in order to gain a genetic template of age- and behavior-associated changes in the brain. In this invention, a validated animal model of mild cognitive impairment is used to determine modulation of gene expression in certain areas of the brain. As shown in Example 1, data reveal a selective modulation of mRNA expression of three genes (Kv4.2, Kcnip2 and DPP6) in the hippocampus among young, aged impaired rats and aged unimpaired rats. The genes are identified as encoding components of a membrane channel that functionally regulates potassium ion (K ⁺ ) conductance in neurons. Together these three genes regulate the flow of potassium through a transmembrane channel.

[0022] Within the superfamily of voltage gated potassium (Kf ¹ ) channels, there arc four closely related subfamilies (Kvl-Kv4). In comparison to the other Kv subfamily members, Kv4 channels have a highly conserved primary structure and mediate a relatively narrow type of K ⁺ currents. Evidence suggests that Kv4 channels (e.g., Kv4.1, Kv4.2 and Kv4.3) may have some unique electrophysiological properties that distinguish them from the other K ^1" channel subfamilies. Kv4 channels may determine fundamental electrical properties of neurons, since they are found in neurons of both primitive and advanced animals

(see Covarrubias et al. for review). [0023] Kv channels are multimeric complexes and are comprised of three domains in the pore-forming α-subunit known as the voltage sensing domain, pore domain and the Tl domain (see Covarrubias et al. for review). Neuronal Kv4 channels also include two integral auxiliary β-subunits including Dipeptidyl- peptidase-like proteins (DPLP) and members of the K ⁺ channel-interacting-protein (Kcnip) family, which are small molecular weight calcium binding proteins. The function of the β-subunits appears to be production of biochemical changes that modulate voltage dependence and ion channel activation/inactivation (Covarrubias et al.). The role of Kcnip proteins appears to be the production of isoform- dependent effects in Kv4 channel trafficking and gating properties. Han et al. demonstrate direct interaction between Kcnip2 and Kv4.2 proteins. In a series of potassium channel protein truncation experiments, Han et al. show that the C- terminus of Kv4.2 is critical for interaction with Kcnip2, cell membrane dependence and cell membrane expression. Modulation of expression and interactions between multiple components of a key neuronal ion channel may have dramatic effects on nervous system function. Emergent patterns in gene expression alteration are potential targets for therapeutic intervention in cognitive decline. This invention also encompasses methods of modulating functional interactions or protein-protein interactions among the protein components of the potassium channel complex, as well as with other proteins. Alteration of Kv4 channel subunit protein-protein interactions may specifically alter neuronal tissue function specifically in the hippocampus. Such functional interactions among ion channel complex proteins are a potential target for drug development in order to ameliorate or prevent cognitive impairment. [0024] As shown in Example 1 , experiments reveal that aged unimpaired rats demonstrate alteration of expression of mRNAs encoding proteins that form a potassium ion channel complex. Alteration of gene expression of elements of a functional protein complex among young and aged rats suggests a correlation between potassium ion channel function and cognitive function. Functionality of the ion complex may be closely related to the stringency of the protein-protein interactions within the multiplex, as well as protein interactions with other proteins that bind to the complex (i.e., functional interaction). [0025] Aspects of this invention include screening assays for candidate compounds that modulate the function of, the abundance of, and/or the protein- protein interactions among potassium ion channel proteins, including Kv4.2, DPP6, Kcnip2 and homologs thereof in order to determine potential therapeutic compounds for treating impaired cognitive function. Aspects of this invention also include screening assays for candidate compounds that modulate gene expression among potassium ion channel proteins including Kv4.2, DPP6, Kcnip2 and homologs thereof in order to determine potential therapeutic compounds for treating impaired cognitive function. [0026] In one aspect of the invention, cognitive function of young, aged impaired and aged unimpaired rats are behaviorally assessed in a water maze. In one embodiment, rats are taught the location of an escape platform guided by a configuration of spatial cues surrounding the maze. In some embodiments of the invention, the platform is visible while in other embodiments the escape platform is camouflaged. Cognitive ability of the young, aged impaired and aged unimpaired rats arc assessed in a water maze and expressed as a learning index.

[0027] Gene expression in the brains of young, aged impaired and aged unimpaired rats is examined using methods commonly known to those of ordinary skill in the art. In a preferred embodiment, expression of mRNA is measured in the brains of young, aged impaired and aged unimpaired rats using microarray analysis. Linear regression analysis was used to determine relationships between mRNA levels and the learning index in aged rats (See Figures 2-4), or between mRNA levels.

[0028] Ion channel subunit function and/or protein-protein interaction is examined in brain cells in culture and in brain tissue of young, aged impaired and aged unimpaired rats using methods commonly known in the art. In one embodiment, protein-protein interaction is determined by measuring function of the potassium ion channels. In one embodiment, protein-protein interaction is determined by measuring function of Kv4.2, DPP6 and Kcnip2 together as an ion channel. [0029] Gonzalez-Ruiz et al. refer to advances in the understanding of the dynamics of protein binding interfaces and methodological developments in the field of structure-based drug design methods. They may allow application of rational design approaches also for finding protein-protein interaction modulators. Methods of determining protein-protein interaction include, but are not limited to, in silico prediction of protein interactions, yeast two hybrid assays, biomolecular interaction analysis (BIA) with mass spectrometry, and assays with dominant negative or mutated sequences (see Archakov et al. for review).

[0030] Examples of candidate compounds that may modulate the function and/or protein-protein interaction in the potassium channel complex, and also affect potassium channel function and abundance and affect cognitive function, include, but are not limited to dimerization inhibitors, and dimerizers, for example (see Archakov et al. for review). This invention also enables design and discovery of small molecules that can interact with multiprotein complexes and modulate their function (see Fry et al. and Gadek et al., for review). See also Sillerud et al. for a review on the design and structure of peptide and peptido mimetic antagonists of protein-protein interaction.

[0031] In one aspect of the invention, young, aged impaired and aged unimpaired rats are behaviorally assessed in a water before gene expression in the brain is examined. In one embodiment of the invention, tissue from young, aged impaired and aged unimpaired rats is collected and processed in a manner described in the Examples below. In some embodiments, whole brain tissue is collected and processed; in other embodiments, the hippocampus region of the brain is collected and processed in a manner described in the Examples below. [0032] The examples described below revealed a decrease in expression of mRNA of three genes in the C A3 sub-region of the hippocampus of unimpaired aged rats as compared to young and aged impaired: KV4.2 (also known as Kcnd2), Kcnip2 (also known as KCWP2) and DPP6. Together these three genes regulate the flow of potassium through the channel Kv4.2 in neurons. Data suggest a coordinated regulation of three component mRNAs that functionally regulate an ion conductance in neurons. The changes observed across the cluster of genes in aged unimpaired rats indicates that cognitive improvement in aging could is attained by targeting the components or their interactions in aged impaired brains to more closely resemble the function achieved in unimpaired aged brains.

[0033] In one aspect, the present invention provides methods of detection of gene expression modulation related to age. In another embodiment, this invention provides methods of identifying a gene or a plurality of genes associated with cognitive impairment in a mammal. In yet another embodiment, this invention provides methods of determining protein-protein interaction among components of the potassium channel complex in mammalian tissue, mammalian cells or eukaryotic cells. This invention provides screening methods to identify compounds that modulate protein-protein interaction among components of the potassium channel complex or function of the potassium channel complex in mammalian tissue, mammalian cells or eukaryotic cells.

[0034] In one aspect of this invention, mammals are used. "Mammals" encompasses, but is not limited to, humans, non-human primates, equines, sheep, canines, felines, pigs, or rodents (e.g., rat). Preferably, the mammals used in identifying the genes of this invention are rats and most preferably, outbred rat strains.

[0035] Eukaryotic cells can be any species of cell, e.g., human cells, mouse cells, rat cells, yeast cells, Xenopus cells, insect cells and non-human primate cells. The cells can be from any known or established eukaryotic cell line, such as those available from the American Type Culture Collection (ATCC; Manassas, VA.), for example, CHO cells, HEK293 cells or COS7 cells. The cells can be in primary cell culture or from a transformed cell line. The cells may naturally express a particular gene product (i.e., the gene may be wholly endogenous to the cell or multicellular organism) or the cell may be a recombinant cell or transgenic organism comprising one or more recombinantly expressed gene products. The cells may be transfected to express an exogenous gene. Preferably, the cells used in embodiments of this invention are of human origin and more preferably are selected from the group consisting of neuronal cells and glial cells, including but not limited to, oligodendrocytes, astrocytes, microglia, pyramidal cells, granule cells, motoneurons, and Purkinje cells. Methods for culturing neuronal cells are well known see, e.g., Brewer et al., [hippocampal neurons]; Walsh et al., [suprachiasmatic neurons]; Zoran et al., [motoneuronal cultures]. Preferably, the neuronal cell is a hippocampal cell or line derived from the hippocampus or a hippocampal region. More preferably, the cell is selected from a CAl , CA3 or DG hippocampal cell. [0036] "Aged" refers to a mammal advanced in years, preferably in or near the latter third of their average lifespan. For example, an aged human would be fifty or more years of age. An aged rat would be fifteen months of age or more.

[0037] "Young" or "Y" refers to one or more adolescent or adult mammals in or near the first third of their average lifespan, at or after the age of sexual maturity and when the hippocampus is fully mature. For example, a young human would be twenty- five years of age or less. A young rat would be ten months of age or less and preferably at least four months of age.

[0038] "AI" or "aged impaired" refers to one or more aged mammals with cognitive impairment relative to young mammals of the same species. The preferred species used as models of age impairment in this invention is rats. Most preferably, the rat is from an outbred strain. Aged rats naturally segregate into two populations, with about half of the aged rats being Al and about half being aged unimpaired (AU). To identify these populations, the cognitive ability of young and aged rats is measured. Methods for identifying populations of AI rats are known in the art, (e.g., Gallagher and Burwell 1989) and an exemplary method is described in the Examples. A number of methods for assessing rat cognitive ability are also known, e.g., the Barnes circular maze, the radial arm maze, the Morris water maze, delayed alternation (delayed nonmatch-to-sample), novel object recognition, conditioned avoidance, and fear conditioning. [0039] "AU" or "aged unimpaired" refers to one or more aged mammals without cognitive impairment relative to young mammals of the same species. The preferred species used as models of age impairment in this invention is rats. Most preferably, the rat is from an outbred strain. As noted above, aged rats naturally segregate into two populations, with about half of the aged rats being AI and about half being AU. To identify these populations, the cognitive ability of young and aged rats can be measured. Methods for identifying populations of AU rats are known in the art, (e.g., Gallagher and Burwell, 1989). A number of methods for assessing rat cognitive ability are known, e.g., the Barnes circular maze, the radial arm maze, the Morris water maze, delayed alternation (delayed nonmatch- to- sample), novel object recognition, conditioned avoidance, and fear conditioning. [0040] "Hippocampal formation" or "hippocampus" or "hippocampal tissue" refers to the whole or part of the hemispheric structure in the brain folded into the ventromedial surface of the temporal lobe, caudal to the amygdaloid complex. Hippocampal tissue comprises hippocampal cells from, without limitation, the CAl, CA3 and DG subregions. Hippocampal cells include, but are not limited to, pyramidal cells of the CA subregions and granule cells of the DG subregion.

[0041] The method comprises providing three populations of mammals - AI, AU and Y - as defined above. CAl, CA3 and DG hippocampal tissue is prepared from each member of the three populations using conventional techniques.

[0030] For the purposes of this disclosure, the gene and gene products identified as Kv4.2 also encompasses, but is not limited to, genes identified as voltage-gated potassium channel, A-type potassium channel, Shal-related subfamily, member 2 (Kcnd2), Kv4 Potassium Channel, Kv4.3L Potassium Channel, Kv4.3 Potassium Channel, Kv4.1 Potassium Channel and homologues thereof.

[0042] For the purposes of this disclosure, the gene and gene products identified as Kcnip2 also encompasses, but is not limited to, genes identified as KChIP2, Kv channel- interacting protein 2, KCMP2.2, Kv channel-interacting protein 2.2 and homologues thereof.

[0043] For the purposes of this disclosure, the gene and gene products identified as DPP6 also encompasses, but is not limited to, genes or gene products identified as dipeptidylpeptidase 6, dipeptidylpeptidase VI, DPPX, dipeptodyl-peptidase-like- proteins (DPLP), , dipeptidyl aminopeptidase-like protein 6 and homologues thereof.

[0044] "Homologue" refers to a gene that has the same origin and functions in two or more species. Preferably, a mammal's homologue of a gene, as identified by the method of the invention, refers to the mammal's equivalent of a gene identified in another mammalian species, e.g., the genes encoding human and rat growth hormones.

[0045] "Cognitive function" or "cognitive status" refers to any higher order intellectual brain process or brain state, respectively, involved in learning and memory including, but not limited to, attention, information acquisition, information processing, working memory, short-term memory, long-term memory, anterograde memory, retrograde memory, memory retrieval, discrimination learning, decision-making, inhibitory response control, attentional set-shifting, delayed reinforcement learning, reversal learning, the temporal integration of voluntary behavior, and expressing an interest in one's surroundings and self-care.

[0046] "Cognitive impairment" or "CI" or an equivalent construct, such as "impaired cognitive function" or "cognitive decline," refers to a deficit or reduction in cognitive status or cognitive function, as defined above, compared to that same function in an age-matched control subject or more usually a population. Cognitive impairment may be observed as a consequence of aging, as well as in various diseases and conditions, including but not limited to, Alzheimer's Disease, Lewy body dementia, vascular dementia, HIV associated dementia, Huntington's Disease, Parkinson's Disease, amyotrophic lateral sclerosis, schizophrenia, depression, MCI, and ARCD. [0047] Cognitive function can be assessed by methods known in the art, for example, a variety of tests known to those skilled in the art can be used to demonstrate cognitive impairment, or the lack thereof, in a human. These tests include, but are not limited to, the Alzheimer's Disease Assessment Scale- cognitive subscale (ADAS-cog), the clinical global impression of change scale (CGIC-plus scale), the Alzheimer's Disease Cooperative Study Activities of Daily

Living Scale (ADCS-ADL), the Mini Mental State Exam (MMSE); the Neuropsychiatric Inventory (NPI), the Clinical Dementia Rating Scale (CDR), the Cambridge Neuropsychological Test Automated Battery (CANTAB), and the Sandoz Clinical Assessment Geriatric (SCAG). In addition, cognitive function may be measured using imaging techniques such as Positron Emission

Tomography (PET), functional magnetic resonance imaging (fMRI), Single Photon Emission Computed Tomography (SPECT), or any other imaging technique that allows one to measure brain activity. In animal model systems, cognitive impairment can be measured in any number of ways known in the art, including using the following apparati: Morris water maze, Barnes circle maze, elevated radial arm maze, T maze or any other mazes in which subjects use spatial information. Other tests known in the art may also be used to assess cognitive impairment, such as fear conditioning, active avoidance, illuminated open-field, dark activity meter, elevated plus-maze, two-compartment exploratory test or forced swimming test.

[0048] "Candidate compound" or "compound" means a pharmaceutical, chemical or other composition of matter with known or unknown physiological effects. A compound can be any natural or synthetic agent made up of one or more elements, including, but not limited to, a small molecule, peptide, polypeptide, peptidomimetic carbohydrate, lipid, protein, glycoprotein, lipoprotein, nucleic acid, and antibody. A compound may or may not have been characterized for its target, or mode of action, in cells or animals prior to its use in the methods of this invention.

[0049] "Beneficially alters" refers to the state of promoting, improving, or preserving cognitive function, or of alleviating or attenuating cognitive decline. A beneficial alteration in accordance with this invention also includes the alleviation or amelioration of one or more manifestations of cognitive impairment, or the delay of onset or slowing of the progression of cognitive impairment. A beneficial alteration of this invention includes, but is not limited to, a change in cognitive function sufficient to result in an improved score in a test of cognitive function; the improvement of cognitive function in a subject with impaired cognitive function so that it more closely resembles the function of a control subject, preferably, e.g., a young subject or an aged unimpaired subject; the improvement over an aged cognitively impaired subject or population; and the preservation of cognitive function over time such that it does not decline or fall below the level observed in the subject upon first presentation or diagnosis. [0050] The methods for measuring the abundance of an expressed gene product according to this invention include, but are not limited to, microarray analysis, macroarray analysis, in situ hybridization histochemistry, fluorescent in situ hybridization (FISH), immunocytochemistry (ICC), immunofluorescence, enzyme linked immunosorbent assay (ELISA), fluorescence polarization immunoassay (FPIA), nephelometric inhibition immunoassay (NIA), immunoprecipitation, quantitative polymerase chain reaction (PCR), RNase protection assay, reverse- transcription PCR, competitive PCR, real-time quantitative PCR (i.e., TaqMan PCR), serial analysis of gene expression (SAGE) analysis, two-dimensional gel electrophoresis, mass spectrometry, MALDI-TOF mass spectrometry, radioimmunoassay (RIA) and blot analysis (e.g. Northern blot, Western blot, protein slot blot, immunoblot, dot blot). If desired, any of the expression and activity assays described above can be used in combination, either sequentially or simultaneously. Such assays can also be partially or completely automated, using methods known in the art.

[0051] A "GCRMA" statistical analysis is an algorithm for deriving gene expression scores from microarrays and preferably GeneChip microarrays. It is an open-source method that is based on robust averaging techniques and sequence- dependent affinity corrections. The robust averaging employed in GCRMA confers a strong immunity to outliers.

[0052] "Age-related cognitive decline" or "ARCD", or an equivalent construct such as "age-associated memory impairment", refers to a diagnosis of mild memory deficit that is not expected to worsen considerably over time. As used herein, ARCD can also be defined as Stage 2 on the Global Deterioration Scale (GDS). The GDS is a seven-point rating system of cognitive and functional capabilities. It is widely used for rating cognitive performance in older adults, with scores ranging from normal aging (Stage 1) to severe dementia (Stage 7). Stage 2 is characterized, for example, by the following clinical characteristics: subjective cognitive complaints in the absence of clinically manifest deficit.

[0053] "Expressed gene product" refers to any form of expression of a gene that can be detected and measured, for example, RNA, amino acid, peptide, polypeptide or protein. It also includes the product itself, whether or not it was actually derived from expression in the cell or tissues of an animal. [0054] "Hippocampus-dependent function" refers to a cognitive function, more specifically a learning or memory process that includes the encoding and acquisition of memories for specific facts and events and episodes of experiences. Hippocampal-dependent function includes the processing of memory representations and the maintenance of memories. Hippocampal-dependent function in mammals includes, for example, spatial memory acquisition, long-term spatial memory, and spatial memory retrieval. A mammal with impaired hippocampal-dependent function may display, e.g., anterograde amnesia for newly acquired facts and events, including maze-specific information in a spatial water maze.

[0055] "Level of cognitive impairment" refers to a measure of the degree of cognitive impairment observed in a mammal. In humans, the level of cognitive impairment may be measured by various neuropsychological tests, alone or in combination, including, but not limited to, the Alzheimer's Disease Assessment Scale-cognitive subscale (ADAS-cog); Global Deterioration Scale (GDS); the clinical global impression of change scale (CIBIC-plus scale); the Alzheimer's Disease Cooperative Study Activities of Daily Living Scale (ADCS-ADL); the Mini Mental State Exam (MMSE); the Neuropsychiatric Inventory (NPI); the Clinical Dementia Rating Scale (CDR); the Rey Auditory Verbal Learning Test (AVLT), Logical Memory Subtest of the revised Wechsler Memory Scale (WMS-

R); the New York University (NYU) Paragraph Recall Test the Cambridge Neuropsychological Test Automated Battery (CANTAB) or the Sandoz Clinical Assessment-Geriatric (SCAG).

[0056] In non-human mammalian models, for example, a rat or non-human primate model, the level of cognitive function may be measured by methods including, but not limited to, using a maze in which subjects use spatial information (e.g., Morris water maze, Barnes circular maze, elevated radial arm maze, elevated plus maze, T- maze and others), recognition tests using odor and novel objects, conditioning tests (e.g., fear conditioning, discrimination tasks, active avoidance, illuminated open-field, two-compartment exploratory test, second and third order conditioning tasks), and tests of higher level executive function (e.g., serial reaction time tests, delayed match and non-match to sample, and stimulus-reward associations including choices involving delayed reinforcement).

[0057] In addition, the level of cognitive function may be measured in mammals, including humans, using neuroimaging techniques, e.g., Positron Emission Tomography (PET), magnetic resonance imaging (MRI), functional magnetic resonance imaging (fJVIRI), Single Photon Emission Computed Tomography (SPECT), or any other imaging technique that allows one to measure brain function. The level of cognitive function in aging can be tested by any of the above methods using aged mammals. [0058] "Mild Cognitive Impairment" or "MCI" refers to a condition characterized by isolated memory impairment accompanied by no other cognitive abnormality and relatively normal functional abilities. One set of criteria for a clinical characterization of MCI specifies the following characteristics: (1) memory complaint (as reported by patient, informant, or physician), (2) normal activities of daily living (ADLs), (3) normal global cognitive function, (4) abnormal memory for age (defined as scoring more than 1.5 standard deviations below the mean for a given age), and (5) absence of indicators of dementia (as defined by DSM-IV guidelines). Petersen et al., Srch. Neurol. 56: 303-308 (1999); Petersen, "Mild cognitive impairment: Aging to Alzheimer's Disease." Oxford University Press, N. Y. (2003).

[0059] Diagnosis of MCI usually entails an objective assessment of cognitive impairment, which can be garnered through the use of well-established neuropsychological tests, including the Mini Mental State Examination (MMSE), the Cambridge Neuropsychological Test Automated Battery (CANTAB) and individual tests such as Rey Auditory Verbal Learning Test (AVLT), Logical

Memory Subtest of the revised Wechsler Memory Scale (WMS-R) and the New York University (NYU) Paragraph Recall Test. See Folstein et al., J Psychiatric Res 12: 189-98 (1975); Robbins et al., Dementia 5: 266-81 (1994); Kluger et al. , J Geriatr Psychiatry Neurol 12:168-79 ( 1999). "Non-human animal model" refers to a non-human mammal or other animal or organism useful for research. [0060] "Increase" in expression refers to an abundance of an expressed gene product that is higher than the abundance of that same product under other conditions or in other cells or tissues. Increased expression may be effected, for example, by one or more structural changes to the gene's encoding nucleic acid or encoded polypeptide sequence (e.g., primary nucleotide or amino acid changes or post- transcriptional modifications such as phosphorylation), altered gene regulation (e.g., in the promoters, regulators, repressors or chromatin structure of the gene), a chemical modification, an altered association with itself or another cellular component, an altered subcellular localization, a modification which causes higher levels of activity through association with other molecules in the cell (e.g., attachment of a targeting domain) and the like.

[0061] "Significant" refers to a confidence level for a measurement being real as opposed to being due to chance, for example, as the result of a random sampling error. In accordance with this invention, significant means a confidence or significance level of at least 95%, wherein p<0.05. More preferably, a significance level of 99%, wherein p<0.01.

[0062] "Decrease" in expression refers to an abundance of an expressed gene product that is lower than the abundance of the same product under other conditions or in other cells or tissues. Such decreased expression may be effected, for example, by one or more structural changes to the gene's encoding nucleic acid or polypeptide sequence (e.g., primary nucleotide or amino acid changes or post- transcriptional modifications such as phosphorylation), altered gene regulation (e.g., in the promoters, regulators, repressors or chromatin structure of the gene), an altered structure (which causes reduced levels of activity), an altered association with itself or another cellular component, an altered subcellular localization, a modification which causes reduced levels of activity through association with other molecules in the cell (e.g., binding proteins which inhibit activity or sequestration) and the like.

[0063] This invention provides methods for identifying compounds useful for treating cognitive impairment by measuring, in a eukaryotic cell, a mammalian cell or a mammalian hippocampus that expresses a potassium channel or the protein subunit thereof in a functional form, the function of a potassium channel or a protein subunit thereof in the presence or absence of a candidate compound and identifying a compound from among the candidate compounds that significantly attenuates the function of the potassium channel or the protein subunit in the mammalian or eukaryotic cell or in the hippocampus of the mammal. An additional embodiment of this invention includes methods for identifying compounds useful for treating cognitive impairment by measuring, in a eukaryotic cell, a mammalian cell or a mammalian hippocampus that expresses a potassium channel or the protein subunit thereof, the abundance of a potassium channel or a protein subunit thereof in the presence or absence of a candidate compound and identifying a compound from among the candidate compounds that significantly reduces the abundance of the potassium channel or the protein subunit in the mammalian or eukaryotic cell or in the hippocampus of the mammal. An addition embodiment of this invention includes methods for identifying compounds useful for treating cognitive impairment by measuring, in a eukaryotic cell, mammalian cell or a mammalian hippocampus that expresses a potassium channel or the protein subunit thereof, the abundance of a mRNA encoding a potassium channel subunit in the presence or absence of a candidate compound and identifying a compound from among the candidate compounds that significantly reduces the abundance of said mRNA, in the mammalian or eukaryotic cell or in the hippocampus of the mammal.

[0064] Preferably, the hippocampal tissue is the CAl, CA3 or DG hippocampal tissue. The same method may also be used for identifying compounds useful in treating age-related cognitive impairment by testing the candidate compounds in aged mammals. In certain embodiments, the potassium ion channel is a voltage- gated potassium ion channel. In certain embodiments, the voltage-gated potassium ion channel is an A-type potassium ion channel. In certain embodiments, the A- typc potassium ion channel is a Shal potassium ion channel. In certain embodiments, the Shal potassium ion channel is a Kv4, Kv4.1, Kv4.2, or Kv4.3 channel. In certain embodiments, the potassium ion channel subunits include Kv4.2, DPP6, Kcnip2 and homologs thereof. In certain embodiments, the eukaryotic cell is from any known or established cell line, such as, without limitation, CHO cells, HEK293 cells or COS7 cells, a primary cell culture, or a transformed cell line. In certain embodiments, the cells naturally express a particular gene product (i.e., the gene may be endogenous or exogenous), a recombinant cell, a cell from a transgenic organism comprising one or more recombinantly expressed gene products. In certain embodiments, the cell is transfected to express an exogenous gene (e.g., an exogenous voltage-gated potassium ion channel or protein subunits thereof). In certain embodiments, the cell is an oocyte. In certain embodiments, the cells used in embodiments of this invention are of human, rat or mouse, Xenopus (e.g., X. laevis) or insect origin. In certain embodiments, the cells are selected from the group consisting of neuronal cells and glial cells, including, but not limited to, oligodendrocytes, astrocytes, microglia, pyramidal cells, granule cells, motoneurons, and Purkinje cells. Preferably, the neuronal cell is a hippocampal cell or line derived from the hippocampus or a hippocampal region, for example, CAl, CA3 or DG regions. In certain embodiments, the hippocampal tissue of a mammal is an intact hippocampus in vivo, an acute hippocampal tissue slice or a hippocampal slice cxplant culture. In certain embodiments, ion channel function is measured clectrophysio logically, as known in the art. Methods for measuring protein and mRNA expression or abundance are well known in the art. In certain embodiments, protein abundance is cell-surface expression. Preferably, compounds useful for treating cognitive impairment are those which alter, enhance or attenuate the functional interaction of potassium ion channel proteins. More preferably, compounds useful for treating cognitive impairment are those which alter, enhance or attenuate the functional interaction of proteins known as Kv4.2, DPP6 and Kcnip2 or their homologs. Preferably, the compound crosses the blood- brain barrier. More preferably, the compound is orally bioavailable.

[0065] An additional embodiment of this invention includes methods for identifying a compound useful in the treatment of cognitive impairment by determining the cognitive status of a mammal in the presence or absence of a candidate compound believed to, or known to, attenuate the function or reduce the abundance of a potassium channel or a protein subunit thereof, and identifying a compound from among the candidate compound(s) that beneficially alters the cognitive status of the mammal with cognitive impairment. In certain embodiments, the mammal is aged. In certain embodiments, the mammal is aged impaired. In certain embodiments, the mammal is a rat. In certain embodiments, the potassium channel is a voltage-gated potassium channel. In certain embodiments, the voltage-gated potassium channel is selected from the group consisting of an A-type potassium channel, a Shal potassium channel, a Kv4 potassium channel, a Kv4.1 potassium channel, a Kv4.2 potassium channel and a Kv4.3 potassium channel. In certain embodiments, the potassium ion channel is a voltage-gated potassium ion channel. In certain embodiments, the voltage-gated potassium ion channel is an A-type potassium ion channel. In certain embodiments, the A-type potassium ion channel is a Shal potassium ion channel.

In certain embodiments, the Shal potassium ion channel is a Kv4, Kv4.1, Kv4.2, or Kv4.3 channel. In certain embodiments, the potassium ion channel subunits include Kv4.2, DPP6, Kcnip2 and homologs thereof. Preferably, compounds useful for treating cognitive impairment are those which alter, enhance or attenuate the functional interaction of potassium ion channel proteins. More preferably, compounds useful for treating cognitive impairment arc those which alter, enhance or attenuate the functional interaction of proteins known as Kv4.2, DPP6 and Kcnip2 or their homologs. Preferably, the compound crosses the blood-brain barrier. More preferably, the compound is orally bioavailable. [0066] An additional embodiment of this invention includes methods for identifying a compound useful in the treatment of cognitive impairment by determining the cognitive status of a mammal in the presence or absence of a candidate compound believed to, or known to, change the functional interactions of at least two expressed gene products of at least two genes identified by the method of this invention, or their homologucs, and identifying a compound from among the candidate compounds that beneficially alters the cognitive status of the mammal. The same method may also be used to identify compounds useful for treating age-related cognitive impairment by testing candidate compounds in aged mammals. Preferably, compounds useful for treating cognitive impairment are those which alter, enhance or attenuate the functional interaction of potassium ion channel proteins. More preferably, compounds useful for treating cognitive impairment are those which alter, enhance or attenuate the functional interaction of proteins known as Kv4.2, DPP6 and Kcnip2 or their homologs.

[0067] As used herein, a compound that is identified and selected from among one or more candidate compounds as being useful in treating cognitive impairment refers to a compound that is capable of altering the abundance or the functional interactions of the expressed gene products of at least one gene identified in accordance with this invention, including potassium ion channel subunit proteins. More preferably, compounds useful for treating cognitive impairment are those which alter the functional interaction of proteins known as Kv4.2, DPP6 and Kcnip2 or their homologs, i.e., in a manner consistent with the beneficial treatment of cognitive function or that is capable of beneficially altering the cognitive status of a mammal to whom it is administered. Preferably, the compound crosses the blood-brain barrier. More preferably, the compound is orally bioavailable.

[0068] A number of different screening protocols can be employed to identify compounds that alter the function or interactions of at least one expressed gene product of a gene or a plurality of genes identified as described above or individually. In general teπns, the methods include culturing cells in the presence of individual members of one or more of candidate compounds to identify a compound that significantly changes in the appropriate direction the abundance of an expressed gene product of a gene or a plurality of genes identified as described including those genes which encode subunits that comprise potassium channel complexes.

[0069] In contrast, a compound that decreases the abundance or attenuates the function of expressed gene products of at least one gene identified in the methods of this invention that was decreased in the AU populations as compared to the combined AI and Y populations would be useful in treating cognitive impairment. Similarly, a compound that increases the abundance or enhances the function of one or more expressed gene products of at least one gene identified in the methods of this invention that was increased in the AU population as compared to the combined AI and Y populations would be useful in treating cognitive impairment.

One or more compounds from among the candidate compounds are then selected based on the compound's ability to significantly change, in the appropriate direction, as defined herein, the abundance or function of the expressed gene product of a selected gene or plurality of genes in the cell treated with the compound.

[0070] Compounds are selected that significantly change _, the abundance or function of the expressed gene product of a gene or plurality of genes selected in the method described above in an aged mammal with cognitive impairment who has been treated with the compound, relative to a member of the group consisting of an aged mammal without cognitive impairment, a young mammal, an aged cognitively impaired mammal in the absence of the compound, and two or more of them. Preferably, the selected compound does not alter that abundance or function in a young mammal and/or an aged cognitively unimpaired mammal to whom the candidate compound is administered. Also preferably, the selected compound does not significantly alter the cognitive status of a young mammal and/or an aged cognitively unimpaired mammal to whom the candidate compound is administered.

[0071] The methods of this invention for identifying compounds useful in the treatment of cognitive impairment, and preferably that due to aging, also include assays based on the cognitive status of a mammal in the presence and absence of the candidate compound. The cognitive status of a mammal, preferably an aged mammal and most preferably an aged cognitively impaired mammal, and more preferably a rat, is determined in the presence and absence of a candidate compound. The cognitive status of the mammal may be assessed using various functions. Preferably, the functions are hippocampal-dependent functions. More preferably, the functions are spatial memory acquisition, long-term spatial memory, and spatial memory retrieval. It is preferable that the candidate compound be one that is believed to, or known to, modulate the abundance or function or functional interactions of an expressed gene product of a gene or plurality of genes encoding components of a potassium ion channel.

[0072] Some embodiments may be based on actual experimental data using other elements of this invention or may be based on information in the scientific literature. A compound that beneficially alters the cognitive status of the treated animal is then selected as being useful in the treatment of cognitive impairment. Preferably, the selected compound beneficially alters the cognitive status in the treated mammal relative to a member of the group consisting of an aged mammal without cognitive impairment, a young mammal, an aged cognitively impaired mammal to whom the compound is not administered and two or more of them. Preferably, the compound does not significantly alter the cognitive status of young mammals or aged unimpaired mammals of the same species that are treated with the compound.

[0073] Another embodiment of the invention is a diagnostic and/or prognostic method. In this embodiment, a mammalian subject that is at risk for developing cognitive impairment or is suspected of having cognitive impairment is tested for changes in levels of expression of genes or gene products associated with age- related cognitive impairment. In some aspects, identification of significant alterations in expression or activity of genes or gene products associated with age- related cognitive impairment is indicative that a mammalian subject has developed age-related cognitive impairment or is at significant risk for development of age- related cognitive impairment as compared to a mammalian subject that does not display significant alterations in expression or activity of genes or gene products associated with age-related cognitive impairment.

[0074] In a preferred embodiment, age-related cognitive impairment is diagnosed or prognosed in a mammalian subject suspected of having agc-relatcd cognitive impairment comprising the steps of obtaining at least one sample of body fluid or tissue from the mammalian subject and also from a control mammalian subject. The body fluid or tissue samples are processed in a manner sufficient to produce a quantifiable amount of at least one gene or gene product associated with age- related cognitive impairment. In a preferred embodiment, the genes or gene product associated with age-related cognitive impairment are at least one member selected from the group of DPP6, Kv4.2, and Kcnip2. The expression or activity level of the at least one gene or gene product associated with age-related cognitive impairment is determined using methods routinely used by those of skill in the art. In some embodiments, the activity level of the gene product includes functional interactions between other gene products, including protein-protein interactions. In some cases, functional interactions among the gene products are required for function of a complex formed by the gene products. In some embodiments, the complex is an ion channel complex. In certain aspects of the invention, the expression or activity level of the at least one gene or gene product associated with age-related cognitive impairment is compared between the sample from a mammalian subject suspected of having age-related cognitive impairment or at risk for development of age-related cognitive impairment and the control. In a preferred embodiment, the genes or gene product associated with age-related cognitive impairment are at least one member selected from the group of DPP6, Kv4.2, and Kcnip2. A significant variation in expression or activity level compared with that of a normal control indicates a diagnosis of age-related cognitive impairment or is prognostic of development of age-related cognitive impairment.

[0075] An additional embodiment to this invention provides a screen for compounds or agents that modulate the expression of KV4.2, Kcnip2 and/or DPP6. In one contemplated embodiment of the invention, a library of candidate compounds is screened for the ability to alter expression of genes in the hippocampus of young, aged impaired and aged unimpaired mammals. In another embodiment, a library of candidate compounds is screened for the ability to alter expression of potassium channel genes in the hippocampus of young, aged impaired and aged unimpaired mammals. In a preferred embodiment, a library of candidate compounds is screened for the ability to alter expression of K.V4.2, Kcnip2 and/or DPP6 genes or gene products.

[0076] An additional embodiment to this invention provides methods and compositions for improving age-related cognitive function and/or treating disorders involving age-related cognitive dysfunction, age-related cognitive impairment or the risk thereof in a subject in need thereof by administering a compound that decreases the abundance or attenuates the function of a voltage-gated potassium channel or a protein subunit thereof (a Kv inhibitor).

[0077] In certain embodiments, the voltage-gated potassium channel is an A-type potassium channel, a Shal potassium channel, a Kv4.1 potassium channel, a Kv4.2 potassium channel or a Kv4.3 potassium channel. In certain embodiments, the protein subunit of a voltage-gated potassium channel is Kv4.2, DPP and Kcnip2. The compound is preferably a compound that alters, enhances or attenuates the functional interaction of potassium ion channel proteins, and more preferably a compound that alters, enhances or attenuates the functional interaction of proteins selected from the group consisting of Kv4.2, DPP6 and Kcnip2.

[0078] In certain embodiments, the age-dependent cognitive impairment is Mild Cognitive Impairment (MCI), Age-Related cognitive Decline (ARCD) or Age- Associated Memory Impairment (AAMI). The subject may be a human or other mammal such as a non- human primate, or rodent (e.g., rat). In one embodiment, the subject is a human patient.

[0079] Candidate compounds for use in the methods of the invention include a wide variety of general types of well-known and available compounds including, but not limited to, small organic molecules (e.g., MW < 1000, preferably < 500); polypeptides; carbohydrates such as oligosaccharides and polysaccharides; polynucleotides; antibodies, lipids or phospholipids; fatty acids; steroids; amino acid analogs, and peptidomimetics. Candidate compounds can be obtained from libraries, such as natural product libraries and combinatorial libraries. A number of different libraries and collections containing large numbers of natural and synthetic compounds are commercially available and one of ordinary skill in the art would be familiar with methods for preparing and obtaining such libraries. Methods of automating assays are also known that permit screening of several thousands of compounds in a short period.

[0080] In one aspect of the invention, an amount of at least one candidate compound is administered to populations of mammals characterized as young, aged impaired and aged unimpaired. The candidate compounds are administered in a sufficient amount and for sufficient time to reasonably expect alteration in gene expression or protein-protein interactions in the brain. In certain aspects of the invention, candidate compounds are administered to rats for a period of time before they are taught to locate an escape platform in a water maze. In other aspects of the invention, candidate compounds are administered to rats at the time they are being taught to locate an escape platform in a water maze. In still another aspect of the invention, the candidate compound is administered to the animals both for a period of time before they are taught to locate an escape platform in a water maze and at the time they are being taught to locate an escape platform in a water maze. [0081] Compounds identified in accordance with the methods of this invention are useful in the treatment of cognitive impairment in mammals, preferably human, and preferably in methods of treating cognitive impairment in aged mammals, preferably humans. The particular compound from among the compounds selected by the methods of this invention to be used can be determined by those skilled in the art, and will depend, for example, on factors such as the severity of the cognitive impairment; the time period over which treatment of the cognitive impairment is desired; whether the compound is administered in a clinical setting or by the individual; or whether an individual suffers from age-related cognitive impairment. [0082] In certain embodiments of the methods of treating cognitive impairment of the invention, the compound useful in treating cognitive impairment is Heteropodatoxin-1, Heteropodatoxin-2, Heteropodatoxin-3 or alpha-KTxl5.

[0083] Compounds, selected in accordance with this invention for use in treating cognitive impairment, and particularly that due to aging, or for use in the screening methods of this invention, can be formulated in pharmaceutical compositions in such a manner to ensure proper distribution in vivo. For example, the blood-brain barrier excludes many highly hydrophilic compounds. To ensure that the selected compounds the blood-brain barrier, they can be formulated, for example, in liposomes, or chemically derivatized. A wide variety of carriers can be used to facilitate targeted drug delivery across the blood-brain barrier to brain tissues, preferably the hippocampus, including but not limited to the use of liposomes, nanoparticles, microparticles, microspheres, encapsulated microbubbles or similar structures which envelope biologically or pharmaceutically active agents, carrier molecules including polymers, and protein including hydrophile proteins. [0084] Administration of a selected compound or candidate compound(s) to be screened can be in a single dose, or in multiple doses. An effective amount of a compound can be determined by those skilled in the art, and can depend on the chemical and biological properties of the compound and the method of contacting the subject. Typically between 0.1 and 1000 mg/kg is administered daily, for one or more days. [0085] In some embodiments, more than one candidate compound is administered in combination. As used herein, administration "in combination" includes simultaneous administration and/or administration at different times, such as sequential administration. The term "simultaneous administration," as used herein, means that the candidate compounds are administered with a time separation of no more than about 15 minutes, such as no more than about 10 minutes. Simultaneous administration of agents encompasses administration as co- formulation or, alternatively, as separate compositions.

[0086] The term "sequential administration" as used herein means that the candidate compounds are administered with a time separation of more than about 15 minutes, such as more than about one hour, or up to 12 hours. Either candidate compound may be administered first. The candidate compounds for sequential administration are contained in discrete dosage forms, optionally contained in the same container or package.

[0087] The candidate compounds can be administered to a subject via any suitable route or routes. Embodiments of this invention include agents administered orally, intravenously, subcutaneously, intra-arterially, intramuscularly, intraspinally, rectally, intrathoracically, intraperitoneally, intracentricularly, or transdermally, topically, or by inhalation. They can be administered orally, for example, in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, or the like, prepared by art recognized procedures.

[0088] When a solid carrier is used for administration, the preparation may be in a tablette, placed in a hard gelatine capsule in powder or pellet form, or it may be in the form of a troche or lozenge. If a liquid carrier is used, the preparation may be in the forms of a syrup, emulsion, soft gelatine capsule or sterile injectable liquid such as an aqueous or nonaqueous liquid suspension or solution. [0089] Test animals will be behaviorally assessed in a preferred embodiment using a water maze. In one embodiment, after behavioral assessment the rats will be sacrificed and dissected as described in the Examples below. Behavior data will be analyzed to determine whether there is any alteration in performance in the water maze in rats which have been exposed to the candidate compound(s). The hippocampal region will be dissected and processed to produce genetic material suitable for gene expression analysis. In a preferred aspect, RNA will be prepared from the rat hippocampal tissue and used in a microarray analysis.

[0090] Gene expression data will be collected and compared among young, aged impaired and aged unimpaired rat that have been treated with at least one candidate compound and young, aged impaired and aged unimpaired rat that have not been treated with at least one candidate compound. Those candidate compounds which significantly alter performance in behavioral tests and expression of genes and gene products in aged impaired animals may be candidates for development into therapeutic compounds.

[0091] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes can be made and equivalents can be substituted without departing from the scope of the invention. In addition, many modifications can be made to adapt a particular situation, material, composition of matter, process, process step or steps, to achieve the benefits provided by the present invention without departing from the scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

[0092] All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an indication that any such document is pertinent prior art, nor does it constitute any admission as to the contents or date of the same. [0093] It will be understood by one of ordinary skill in the art that the compositions and methods described herein may be adapted and modified as is appropriate for the application being addressed and that the compositions and methods described herein may be employed in other suitable applications, and that such other additions and modifications will not depart from the scope hereof.

[0094] This invention is illustrated by the following examples, which are not to be construed as imposing limitations on the scope thereof. On the contrary, various other embodiments, modifications, and equivalents thereof, which after reading the description herein, may suggest themselves to those skilled in the art, may be made without departing from the spirit or scope of the present invention.

EXAMPLES

[0095] Preparation of RNA from Rat tissue

[0096] Twenty- four outbred Long-Evans rats were killed by live decapitation to obtain fresh brain tissue. The brain was removed and hippocampus region was dissected. There were ~8 animals in each group (AI, AU and Y). [0097] Total RNA was isolated using Trizol reagent (INVITROGEN™, Carlsbad, CA) according to the standard protocol (homogenization in Trizol reagent followed by chloroform extraction and isopropanol precipitation). Total RNA was further purified using the RNeasy mini kit (QIAGEN™, Valencia, CA). cRNA probes were then generated from the RNA samples at The Johns Hopkins Microarray Core Facility, generally according to Affymetrix specifications as detailed herein.

[0098] Briefly, 5 micrograms of total RNA were used to synthesize first strand cDNA using oligonucleotide probes with 24 oligo-dT plus T7 promoter as primer (Proligo LLC, Boulder, CA) , and the Superscript ChoiceSystem (INVITROGEN™). Following the double stranded cDNA synthesis, the product was purified by phenol-chloroform extraction, and biotinylated anti-sense cRNA was generated through in vitro transcription using the BioArray RNA High Yield Transcript Labeling kit (ENZO Life Sciences Inc., Farmingdale, NY). Fifteen micrograms of the biotinylated cRNA was fragmented at 94°C for 35 min (10OmM Trix-acetate, pH 8.2, 500 mM KOAc, 150 mM MgOAC). lOμg of total fragmented cRNA was hybridized to the RAT genome 230-2 Affymetrix GeneChip array for 16 hours at 45°C with constant rotation (60 rpm). The cRNA prepared from the CAl , C A3 or DG regions for each animal were hybridized to an individual microarray. |0099] Affymetrix Fluidics Station 450 was then used to wash and stain the chips, removing the non-hybridized target and incubating with a streptavidin- phycoerythrin conjugate to stain the biotinylated cRNA. The staining was then amplified using goat immunoglobulin-G (IgG) as blocking reagent and biotinylated anti-streptavidin antibody (goat), followed by a second staining step with a streptavidin-phycoerythrin conjugate.

[0100] For quality control of the total RNA from the samples, the Agilent Bioanalyzer Lab on a Chip technology, was used to confirm that all the samples had optimal rRNA ratios (1 :2, for 18S and 28S, respectively) and clean run patterns. For quality control of the hybridization, chip image, and comparison between chips, the following parameters were considered: Scaling factor: related to the overall intensity of the chip, to confirm the similar signal intensity and staining throughout the samples; Background: estimation of unspecific or cross- hybridization; Percentage of present calls (for "MAS5" analysis only, see infra, Data Analysis of Microarray): percentage of transcripts that are considered significantly hybridized to the chip (present) by the algorithm; Glyceraldehydc-3- phosphate dehydrogenase (GAPDH) (375'): representation of the RNA integrity by measuring the ratio of 3' to 5' regions for the housekeeping gene GAPDH, its presence in the chip and a ratio close to 1 advocates for a good integrity of the target (sample); Spikes (BioB/BioC) to confirm the detection level and sensitivity after hybridization.

[0101] Data Analysis of Microarray. Data were obtained using Affymetrix Gen Chip Rat Expression Set 230 for the dissected CA3 region of the hippocampus. Fluorescence was detected using the Affymetrix G3000 GeneArray Scanner and image analysis of each GeneChip was done through the GeneChip Operating System 1.1.1 (GCOS) software from Affymetrix, using the standard default settings. All of the GeneChip arrays use short oligonucleotides to probe for genes in an RNA sample. [0102] The method used to process and summarize the probe set data was "GCRMA" analysis. This statistical method employs background adjustment by estimating a global signal over the entire probe set and making a whole array adjustment, using quantile normalization. GCRMA is an open-source method that is based on robust averaging techniques and sequence- dependent affinity corrections. The robust averaging employed in GCRMA confers a strong immunity to outliers (See Wu et al.).

[0103] EXAMPLE 1 : Microarray Analysis of Gene Expression in Mammalian Hippocampus. [0104] The following data were obtained using Affymetrix GeneChip Rat Expression Set 230 for the dissected CA3 region of the hippocampus. After extensive microarray quality assessment, data were analyzed for 8 young, 8 aged impaired and 7 aged unimpaired rats. Multidimensional scaling eliminated two additional outlier AU chips to yield N=5 for aged unimpaired. Probe set intensities were normalized using GCRMA and statistical analysis performed on these data using the Significance Analysis in Microarrays.

[0105] An ANOVA was conducted on the probe set signal values for all present probe sets by combining two groups of animals and comparing them to the third group. An "AU ANOVA" was performed, where Aged Impaired were combined with Young and compared to Aged Unimpaired. Any probe sets having a p-value less than 0.05 were considered significantly changed and the genes or plurality of genes selected.

[0106] Pearson's correlations comparing probe set signal values to learning indices were then calculated for the aged animals (excluding young) across all present probe sets. Again, correlations representing a p-value of less than 0.05 were considered significantly changed.

Comparison of gene expression levels in the dorsal hippocampus among young, aged unimpaired rats and aged impaired rats showed that gene expression levels of Kv4.2, Kcnip2 and DPP6 in aged unimpaired (AU) rats were significantly lower than in young (Y) and aged impaired (AI) rats. See Figures 1-4. Linear regression analysis of the relationship between mRNA levels of Kv4.2, Kcnip2 and DPP6 and the learning index between aged-impaired animals (♦ filled diamond) and aged- unimpaired animals (0 empty diamond) also showed that lower expression of Kv4.2, Kcnip2 and DPP6 in aged rats correlated to better performance in the spatial learning task. The finding of low expression of Kv4.2 in aged unimpaired (AU) rats compared to young (Y) and aged impaired (AI) rats was confirmed at the level of protein expression by immunohistochemistry. See Figure 5. Linear regression analysis of the relationship between protein levels of Kv4.2 and the learning index between aged-impaired animals (A filled triangle) and aged-unimpaired animals (■ filled square) also showed that lower expression of Kv4.2 in aged rats correlated to better performance in the spatial learning task. Comparing Kv4.2 expression to distance from the soma showed that a significant reduction of Kv4.2 protein expression in AU rats was found at distal dendritic regions nearing the perforant path input from the entorhinal cortex, within both the CA3 stratum radiatum and stratum lacunosum (Figure 6C).

[0107] The findings reported here will also be validated using other methods, including in situ hybridization histochemistry to determine amount and cellular localization of mRNA changes in the hippocampus. Compounds will be tested for ability to modify target mRNAs and protein expression in the test system. [0108] However, the instant data also indicate that modulation of a protein system or of the interaction of units in a protein system is a viable target for drug discovery relating to cognitive impairment in aging. Methods of identifying protein-protein interacting surfaces, and drug design based upon such identified surfaces, are included in the illustrative, but non-limiting, references below. For example, it can be appreciated by one of skill in the art that these techniques may be adapted to design or identify compounds that modulate the interaction of the KV4.2, DPP6, Kcnip2, and Kv4.2 channel, with each other(s) (such as the interaction of KV4.2 and Kcnip2 disclosed in Han et al.) and different protein partners. Drugs which reduce functionality of such protein functional units may be useful in treatment of cognitive impairment in aging. [0109) EXAMPLE 2 : Identification of a compound that modulates protein protein interactions in vitro.

[0110] Identification of a compound useful in the treatment of cognitive impairment in aging by modulation of the interaction of at least two functionally interactive proteins with roles in cognitive function is performed in the following manner. At least two proteins known to functionally interact with one another are mixed in vitro under physiological conditions conducive to interaction of the proteins.

[0111] This example is performed in a mammalian or eukaryotic cell lysate containing Kv4.2, DPP6 and Kcnip2 prepared and kept under appropriate physiological conditions to allow functional interaction of the protein components. In this example, the proteins include Kv4.2, DPP6 and Kcnip2. A compound that is a candidate for significantly altering the interaction of Kv4.2, DPP6 and Kcnip2 is selected from a library of compounds. An assay for functional interaction of Kv4.2, DPP6 and Kcnip2 is performed in the presence and absence of a sufficient amount of the candidate compound to measurably alter the functional interactions of Kv4.2, DPP6 and Kcnip2. Interaction of Kv4.2, DPP6 and Kcnip2 are quantitated in the presence and absence of the candidate compound. Differences in the quantitated amounts of functional interaction of the proteins in a mixture of proteins in the presence and absence of the candidate compound are determined. A significant increase or decrease in protein interaction in the presence of the candidate compound is correlated with usefulness in treatment of cognitive impairment in aging.

[0112] EXAMPLE 3: Identification of a compound that is useful in treatment of cognitive impairment with an in vivo protocol.

[0113] Identification of a compound useful in the treatment of cognitive impairment in aging by modulation of the interaction of at least two functionally interactive proteins with roles in cognitive function is performed in the following manner. [0114] Specifically, Kv4 potassium ion channels are of interest. Activity of Kv4 potassium ion channels is measured in a manner commonly performed by those of skill in the art and may include electrophysiological measurements.

[0115] This example is performed in populations of outbred Long Evans rats expressing Kv4.2, DPP6 and Kcnip2. A compound that is a candidate for significantly altering the interaction of Kv4.2, DPP6 and Kcnip2 is selected from a library of compounds. The candidate compound is administered to the rat in a sufficient time and amount to provoke a change in the Kv4 potassium channel activity. [0116] An assay for Kv4 potassium channel activity is performed in the presence and absence of a sufficient amount of the candidate compound. Kv4 potassium channel activity is quantitated in the presence and absence of the candidate compound. Differences in the quantitated Kv4 potassium channel activity in the presence and absence of the candidate compound are determined. A significant reduction in Kv4 potassium channel activity in the presence of the candidate compound is correlated with usefulness in treatment of cognitive impairment in aging.

[0117] EXAMPLE 4: Identification of a compound that is useful in treatment of cognitive impairment with an in vitro protocol. [0118] Identification of a compound useful in the treatment of cognitive impairment in aging by modulation of the interaction of at least two functionally interactive proteins with roles in cognitive function is performed in the following manner.

[0119] Mammalian neuronal cells are isolated and cultured in a manner conducive to function of potassium ion channels present in the membrane of the neuronal cells. Specifically, Kv4 potassium ion channels. Alternatively, eukaryotic cell lines and oocytes expressing Kv4 potassium ion channels or protein subunits thereof are used. The cells may express Kv4 potassium ion channels or protein subunits thereof endogenously, or the cells may be of transgenic origin or be transfected to express exogenous Kv4 potassium ion channels or protein subunits thereof. Activity of Kv4 potassium ion channels is measured in a manner commonly performed by those of skill in the art and may include electrophysiological measurements.

[0120] This example is performed in a culture of mammalian neuronal cells containing Kv4.2, DPP6 and Kcnip2 prepared and kept under appropriate physiological conditions to allow functional interaction of the protein components. In this example, the proteins include Kv4.2, DPP6 and Kcnip2. A compound that is a candidate for significantly altering the interaction of Kv4.2, DPP6 and Kcnip2 is selected from a library of compounds. The candidate compound is applied to the mammalian neuronal cell cultures in a sufficient time and amount to provoke a change in the Kv4 potassium channel activity.

[0121] An assay for Kv4 potassium channel activity is performed in the presence and absence of a sufficient amount of the candidate compound to measurably alter the functional interactions of Kv4.2, DPP6 and Kcnip2. Kv4 potassium channel activity is quantitated in the presence and absence of the candidate compound. Differences in the quantitated Kv4 potassium channel activity in the presence and absence of the candidate compound are determined. A significant reduction in Kv4 potassium channel activity in the presence of the candidate compound is correlated with usefulness in treatment of cognitive impairment in aging.

[01221 Introduction and Models of Age-Related Cognitive Impairment [0123] A variety of conditions characterized by cognitive impairment (e.g., Age Associated Memory Impairment [AAMI], Mild Cognitive Impairment [MCI] and Age-Related Cognitive Decline [ARCD]) are believed to be related to aging. Animal models serve as an important resource for developing and evaluating treatments for such age-related cognitive impairments. Features that characterize age-related cognitive impairment in animal models typically extend to age-related cognitive impairment in humans. Efficacy in such animal models is, thus, predictive of efficacy in humans.

[0124] Of available models, a Long-Evans rat model of cognitive impairment is particularly well suited for distinguishing the difference between cognitive impairment related to illness and that related to aging. Indeed, extensive behavioral characterization has identified a naturally occurring form of cognitive impairment in an outbred strain of aged Long-Evans rats (Charles River Laboratories; Gallagher et al., Behav. Neurosci. 107:618-626, (1993)). In a behavioral assessment with the Morris Water Maze (MWM), rats learn and remember the location of an escape platform guided by a configuration of spatial cues surrounding the maze. The cognitive basis of performance between young rats (6 months of age), and aged rats (26-27 months of age) are tested in probe trials using measures of the animal's spatial bias in searching for the location of the escape platform. Aged rats in the study population have no difficulty swimming to a visible platform, but an age-dependent impairment is detected when the platform is camouflaged, requiring the use of spatial information. Performance for individual aged rats in the outbred Long-Evans strain varies greatly. For example, a proportion of those rats perform on a par with young adults. However, approximately 40-50% fall outside the range of young performance. This variability among aged rats reflects reliable individual differences. Thus, within the aged population some animals are cognitively impaired and designated aged- impaired (Al) and other animals are not impaired and are designated agcd- unimpaircd (AU). See, e.g., Colombo et al., Proc. Natl. Acad. Sci. 94: 14195- 14199, (1997); Gallagher and Burwell, Neurobiol. Aging 10: 691-708, (1989); Rapp and Gallagher, Proc. Natl. Acad. Sci. 93: 9926-9930, (1996); Nicolle et al., Neuroscience 1A: 1A\ -756, (1996); and Nicolle et al., J. Neurosci. 19: 9604-9610, (1999), Gallagher et al. Behav. Neurosci. 107:618-626, (1993).

[0125] We use this rat model described above to identify individual AI and AU rats. We then conduct histological experiments on them, as well as tested their performance in further MWM tasks while administering various pharmacological treatments.

[0126] Example 5: Behavioral assessment of Kv inhibitor treatment in a 6 hour-delay memory task.

Aged rats that demonstrated impaired memory performance in a standardized assessment of spatial cognition (Gallagher et al., 1993), i.e., AI rats, are selected for the drug intervention studies. AI Rats are treated with or a compound that decreases the abundance or attenuates the function of a voltage-gated potassium channel or a protein subunit thereof (a Kv inhibitor). The voltage-gated potassium channel may be an A-type potassium channel, a Shal potassium channel, a Kv potassium channel, a Kv4.1 potassium channel, a Kv4.2 potassium channel or a Kv4.3 potassium channel. The protein subunit of a voltage-gated potassium channel may be Kv4.2, DPP and Kcnip2. The compound is preferably a compound alters the functional interaction of potassium ion channel proteins, and more preferably a compound that alters the functional interaction of proteins selected from the group consisting of Kv4.2, DPP6 and Kcnip2. The compound may be Heteropodatoxin- 1 , Heteropodatoxin-2, Heteropodatoxin-3 or alpha- KTxI 5. The rats are then trained and tested in a new water maze environment. The water maze used is housed in a different building and is surrounded by black curtains with a novel set of white patterns. The training protocol used is based on a modified water maze task known to be highly hippocampal-dependcnt (de Hoz ct al., 2005; Steele & Morris, 1999). Unlike the traditional water maze protocol wherein the escape platform location remained constant throughout training, the escape platform location in this spatial memory version of the task varies from day to day.

[0127] The test is comprised of a training phase followed by a memory retention test. Each day, during the training phase, rats are given six trials to locate the submerged escape platform. On each trial, a rat is released in the maze from one of four equally spaced starting positions around the perimeter of the pool. The starting position varies from trial to trial. If the rat does not locate the escape platform within 60 seconds on any trial, the experimenter guides and places the rat on the platform, where it remained for 20 seconds. The rat is then removed from the platform and placed in a holding cage for another 40 seconds before the next trial. The length of the path the rat took while swimming in the maze is measured in each trial. At the end of the 6 trials of the training session, the rats are returned to their home cages in the colony room for a 6-hour waiting period, in preparation for the subsequent memory retention test.

[0128] After the 6-hr waiting period, the rats are returned to the water maze for a test of spatial memory with the submerged platform located in the same position as in the training trials. Each rat is given 60 second to locate the platform. Rats are trained and tested in this manner for five consecutive days. Performances on the last three days are averaged for analysis. Memory-impaired aged rats treated with either the Kv inhibitor or saline perform at comparable levels at the end of the training phase (Trials 1 -6). However, those treated with the Kv inhibitor shows less forgetting (i.e., memory savings or retention) after a 6-hr delay compared to their counterparts treated with saline.

[0129] Proteins encoded by the following sequences, their homologues and functional equivalents, are included (but not limiting) in the instant invention. [0130] Dipeptidyl-peptidase 6; DPP6

1 : NM_001039350 Homo sapiens dipeptidyl-peptidase 6 (DPP6), transcript variant 3, mRNAgi|86792862|ref]NM_001039350.1|[86792862]

2: NM_001936 Homo sapiens dipeptidyl-peptidase 6 (DPP6), transcript variant 2, mRNA gi|86792777|reflNM_001936.3|[86792777]

3: NM_130797 Homo sapiens dipeptidyl-peptidase 6 (DPP6), transcript variant 1 , mRNA gi|86792773|rcflNM_130797.2|[86792773] 4: BC 150304 Homo sapiens dipeptidyl-peptidase 6, mRNA (cDNA clone MGC: 167068 IMAGE:8860401), complete cds gi| 152012558jgb|BC 150304.11[ 152012558]

5: NM_022850 Rattus norvegicus dipeptidylpeptidase 6 (Dpp6), mRNA gi| 12408297|ref]NM_022850.11[ 12408297] 6: M76426 Rattus norvegicus dipeptidyl aminopeptidase-related protein (dppό) mRNA, complete cds gi|408713|gb|M76426.1 |RATDAPRP[408713]

7: M76427 Rattus norvegicus dipeptidyl aminopeptidase-related protein (dpp6) mRNA, complete cds gi|408715|gb|M76427.1 |RATDAPRPA[408715]

[0131] Potassium voltage gated channel, Shal-related family, member 2; Kcnd2

1 : NM_012281 Homo sapiens potassium voltage-gated channel, Shal-related subfamily, member 2 (KCND2), mRNA gi|27436982|ref)NM_012281.2|[27436982]

2: AJ010969 Homo sapiens mRNA for potassium channel Kv4.2 gi|6006516|emb| AJ010969.1 | [6006516]

3: AFl 21104 Homo sapiens potassium channel KV4.2 (KCND2) mRNA, complete cds gi|4530477|gb|AF121104.1|AF121104[4530477] 4: BCl 10450 Homo sapiens potassium voltage-gated channel, Shal-related subfamily, member 2, mRNA (cDNA clone MGC: 1 19703 IMAGE:40012009), complete cds gi|83405878|gb|BCl 10450.1|[83405878]

5: BCl 10449 Homo sapiens potassium voltage-gated channel, Shal-related subfamily, member 2, mRNA (cDNA clone MGC: 1 19702 IMAGE:40012007), complete cds gi|83405490|gb|BCl 10449.1|[83405490]

6: NM_031730 Rattus norvegicus potassium voltage gated channel, Shal-related family, member 2 (Kcnd2), mRNA gi|76881821|reflNM_031730.2|[76881821]

7: M59980 Rat voltage-gated K ⁺ channel protein (RK5) mRNA, complete cds gi|203467|gb|M59980.1 |RATCK5 A[203467]

8: AF223160 Rattus norvegicus potassium channel Kv4.2 mRNA, 5' UTR and partial cds gi|7021400|gb| AF223160.1 |AF223160[7021400]

[0132] Kv channel-interacting protein 2; Kcnip2

1 :NM_014591 Homo sapiens Kv channel interacting protein 2 (KCNIP2), transcript variant 1, mRNA gi|50557654|ref|NM_014591.4|[50557654]

2: NM_173191 Homo sapiens Kv channel interacting protein 2 (KCNIP2), transcript variant 2, mRNA gi|50557653|ref]NM_173191.2|[50557653]

3: NM 173192 Homo sapiens Kv channel interacting protein 2 (KCNIP2), transcript variant 3, mRNA gi|50557649|ref]NM_173192.2|[50557649] 4: NM 73193 Homo sapiens Kv channel interacting protein 2 (KCNIP2), transcript variant 4, mRNA gi|50557655|ref|NM_173193.2|[50557655]

5: NM_73194 Homo sapiens Kv channel interacting protein 2 (KCNIP2), transcript variant 5, mRNA gi|50557651 |reflNM_173194.2|[50557651]

6: NM_173195 Homo sapiens Kv channel interacting protein 2 (KCNIP2), transcript variant 6, mRNA gi|50557650|ref]NM_173195.2|[50557650]

7: NM_173197 Homo sapiens Kv channel interacting protein 2 (KCNIP2), transcript variant 7, mRNA gi|50557652|reflNM_173197.2|[50557652]

8: AY302141 Homo sapiens Kv channel interacting protein 2 (KCN1P2) mRNA, complete cds; alternatively spliced gi|31747540|gb|AY302141.11[31747540] 9: AJ276317 Homo sapiens partial mRNA for A-type potassium channel modulatory protein 2 (KCHIP2 gene), 3TJTRgi|7228295|emb| AJ276317.11[7228295]

10: AF199598 Homo sapiens A-type potassium channel modulatory protein 2 (KCHIP2) mRNA, complete cds gi|6969256|gb|AFl 99598.1|AF199598[6969256] 1 1 : AF295530 Homo sapiens cardiac voltage gated potassium channel modulatory subunit mRNA, complete cds, alternatively spliced gi|9930624|gb|AF295530.1 |AF295530[9930624]

12: DQ 148484 Homo sapiens potassium channel interacting protein 2 mRNA, complete cds, alternatively spliced gi|73543413|gb|DQ 148484.11[73543413]

13: DQ 148483 Homo sapiens potassium channel interacting protein 2 mRNA, complete cds, alternatively spliced gi|7354341 l |gb|DQ148483.1 |[7354341 1]

14: DQ 148482 Homo sapiens potassium channel interacting protein 2 mRNA, complete cds, alternatively spliced gi|73543409|gb|DQ148482.1 |[73543409] 15: DQ 148481 Homo sapiens potassium channel interacting protein 2 mRNA, complete cds, alternatively spliced gi|73543407|gb|DQ148481.1|[73543407]

16: DQ 148480 Homo sapiens potassium channel interacting protein 2 mRNA, complete cds, alternatively spliced gi|73543405|gb|DQ148480.1|[73543405]

17: DQ862463 Homo sapiens Kv channel interacting protein 2 transcript variant 9 (KCNIP2) mRNA, complete cds, alternatively spliced gi|1 14150679|gb|DQ862463.1 |[l 14150679]

18. AF367021 Homo sapiens KCHIP2.4 (KCNIP2) mRNA, complete cds, alternatively spliced gi| 14091331 |gb| AF367021.11 AF367021 [14091331]

19. AF367020 Homo sapiens KCHIP2.6 (KCNIP2) mRNA, complete cds, alternatively spliced gi| 14091329jgbf AF367020.11 AF367020[ 14091329]

20. AF367019 Homo sapiens KCHIP2.5 (KCNIP2) mRNA, complete cds, alternatively spliced gi| 14091327|gb|AF367019.11 AF367019[ 14091327]

21. AF367018 Homo sapiens KCHIP4.2 (KCN1P2) mRNA, complete cds, alternatively spliced gi|14091325|gb|AF367018.1|AF367018[14091325] 22. AK027347 Homo sapiens cDNA FLJ 14441 fis, clone HEMBB 1000927, highly similar to Homo sapiens A-type potassium channel modulatory protein 2 (KCH1P2) mRNA gi| 14041962|dbj| AK027347.11[ 14041962]

23. AY026328 Homo sapiens Kv channel-interacting protein 2 isoform 2 (KCMP2) mRNA, complete cds, alternatively spliced gi| 13919623|gb| AY026328.11[ 13919623]

24. AF3471 14 Homo sapiens Kv channel interacting protein 2.2 (KCNIP2) mRNA, complete cds; alternatively spliced gi|14586767|gb|AF3471 14.1|AF3471 14[14586767]

25: BC034685 Homo sapiens Kv channel interacting protein 2, mRNA (cDNA clone MGC: 17241 IMAGE:4155871), complete cds gi|21961 182|gb|BC034685.1 |[21961 182] 26. AF295076 Homo sapiens cardiac voltage gated potassium channel modulatory subunit, short form mRNA, partial cds, alternatively spliced gi|9930621 |gb| AF295076.11 AF295076[9930621 ]

27. NM_001033961 Rattus norvcgicus Kv channel- interacting protein 2 (Kcnip2), transcript variant c, mRNA gi|76881829|ref]NM_001033961.11[76881829]

28: NM_020094 Rattus norvegicus Kv channel-interacting protein 2 (Kcnip2), transcript variant a, mRNAgi|76881825|reflNM_020094.2|[76881825]

29: NM 020095 Rattus norvegicus Kv channel-interacting protein 2 (Kcnip2), transcript variant b, mRNA gi|76881827|ref]NM_020095.2|[76881827] 30: AB040031 Rattus norvegicus rKCHIP2a mRNA for A-type potassium channel modulatory protein 2a, complete cds gi|72S9286|dbj| AB040031.11[7259286]

31 : AB040032 Rattus norvegicus rKCHIP2b mRNA for A-type potassium channel modulatory protein 2b, complete cds gi|7259288|dbj| AB040032.11[7259288]

32: AF269283 Rattus norvegicus potassium channel auxiliary subunit KCHIP2a mRNA, complete cds gi|8926236|gb|AF269283.1|[8926236]

33: AF269284 Rattus norvegicus potassium channel auxiliary subunit KCHIP2b mRNA, complete cds gi|8926238|gb|AF269284.1|[8926238]

34: AF269285 Rattus norvegicus potassium channel auxiliary subunit KCHIP2c mRNA, complete cds gi|8926240|gb|AF269285.1|[8926240] 35: BCO859O5 Rattus norvegicus Kv channel-interacting protein 2, mRNA (cDNA clone MGC:94740 IMAGE: 7193750), complete cds gi|55249795|gb|BC085905.11[55249795]

36: NM OO 1034005 Rattus norvegicus A-type potassium channel modulatory protein 2 (Kchip2), rnRNAgi|77404418|ref]NM_001034005.1 |[77404418]

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