CLAIM TO PRIORITY

[0001] This application claims benefit of priority of U.S. Application Serial Number

61/187,061 filed June 15, 2009, and EU Application Serial Number EP10001268 filed February 8, 2010.

FIELD OF THE INVENTION

[0002] The present invention provides an in vitro or cell-based method to identify memory modulating compounds based on the assessment of activity of the Kidney BRAin protein (WWCl or KIBRA) protein. The disclosed methods herein are also suitable to uncover memory related pathways and to test the relevance of memory modulating strategies.

BACKGROUND OF THE INVENTION

[0003] The protein KIBRA (for Kidney BRAin protein, also known as WWC 1) and the KIBRA allele are associated with memory performance and cognition in healthy subjects. Carriers of the KIBRA (rsl7070145) T allele performed better on multiple episodic memory tasks than those homozygous for the C allele at rsl7070145. Additionally, stronger brain activation in key areas of learning and memory can be observed in subjects carrying the C allele in comparison to T allele carriers during a memory task. Activation measured during episodic memory task, using functional magnetic resonance imaging, suggests that non-carriers of the T allele require more activation in memory related brain regions to reach the same retrieval performance as the T allele carriers.

[0004] The association of KIBRA polymorphism with memory performance was confirmed in various studies in healthy subjects and in Alzheimer's disease patients. Aging populations will suffer from an increase of different forms of memory impairments like Alzheimer' s disease, aging related cognitive decline, mild cognitive impairment, or vascular dementia.

BRIEF DESCRIPTION OF THE FIGURES [0005] FIG. 1 depicts the KIBRA-GFP protein alternation in the subcellular distribution in the presence of activated PKC-zeta.

[0006] FIG. 2 depicts the florescence microscopy of the subcellular location of fluorescent KIBRA-EGFP fusion protein expressed in COS cells, in the absence (FIG. 2A) and in the presence (FIG. 2B) of co-expression of myristinylated PKC zeta.

DETAILED DESCRIPTION

[0007] The present disclosure provides an in vitro or cell-based method to identify memory modulating compounds based on the assessment of activity of the Kidney BRAin protein (WWCl or KIBRA) protein. The disclosed methods herein are also suitable to uncover memory related pathways or to test the relevance of memory modulating strategies. The present disclosure provides for assays for use in pharmaceutical research to find new treatment options for multiple neurological conditions where learning and memory is impaired. Thus, the present disclosure paves the way for novel treatment options using KIBRA as a novel pharmacological target to treat multiple forms of memory impairment due to the discovered link to Alzheimer' s disease pharmacological accessibility.

[0008] Moreoever, it should be recognized that the present disclosure provides for carrying out the described methods by adding a molecular entity such as an antibody, gene, small molecule or protein to a cell that contains an expression construct that includes the KIBRA gene and a reporter gene in such a configuration that the KIBRA gene is coexpressed with the reporter gene. A KIBRA-reporter gene fusion is one example of such a configuration. Differential localization (of the reporter in a cell to which the molecular entity was added compared to a control cell that contains the expression construct but to which the molecular entity was not added) correlates with the identification of the molecular entity as having effects upon KIBRA. The punctate nature of the localization then may be quantified. It is recognized that the reporter may be luciferase, GFP, or any other expressable protein. [0009] In fact, the present disclosure demonstrates that the subcellular localization of KIBRA could be used as a readout in a method for identifying memory or cognition enhancing compounds. This is based on the observation that the activation of PKC zeta, which is regarded central for memory related pathways can be measured by the redistribution of KIBRA from a punctuated pattern to a cell membrane associated pattern. PKC zeta activation can be enabled by a myristinylation taq that tracks PKC zeta to the cell membrane, the compartment, where the kinase exerts its function in memory related pathways. The redistribution of KIBRA in cell culture assays can be monitored e.g. by fusion proteins of KIBRA with fluorescent proteins such as "green fluorescent protein" (GFP) or by the immunostaining for KIBRA using appropriated antibodies or by immunostaining for tagged KIBRA proteins using tag-binding antibodies.

[0010] As described further herein, the role of KIBRA for neuronal plasticity is further underlined by the observation that KIBRA interacts with protein kinase C zeta (PKC zeta), a molecule crucially involved in neuronal plasticity. PKC zeta can phosphorlylate KIBRA. PKC zeta, and also protein kinase M zeta (PKM zeta), the constitutively active form of PKC zeta, are both involved in learning and memory, and this kinase influences memory maintenance in animals in the hippocampus and in the cortex. Moreover, specific inhibition of the PKM zeta activity disrupts the maintenance of LTP, a process regarded as a direct correlate of memory, whereas the activation of PKM zeta leads to increased LTP.

[0011] Therefore, the present disclosure provides a method of assessing whether or not a molecular entity affects the protein encoded by the KIBRA gene. The present disclosure further provides a method of screening potential effectors of the KIBRA gene. The present disclosure additionally provides a method to screen compounds as potential treatments of Alzheimer's disease and other neurodegenerative disorders. Finally, the present invention provides for a method to screen compounds as potential augmentors of human memory.

[0012] Accordingly, the term "modulation" or "modulate" means the partial or essentially complete increase or decrease. The terms "compound" and "substance" are to be understood broadly and means, in a general manner, all the material means and substances, which can be contacted to cultured cells, e.g. but without any claim of completeness: low molecular weight compounds, peptides, polypeptides, antibodies, nucleic acids, antisense nucleic acids, SiRNA, aptameres, natural or artificial transcription factors, nucleic acid constructs, vectors, viral constructs, or liposomal means.

[0013] The present disclosure provides for an assay that assesses the activity of

KIBRA protein in response to a molecular entity to be tested with regard to its effect on

KIBRA. A molecular entity may be any molecule or chemical element that may be added to a cell in such a way that it has the opportunity to affect a biological activity. Molecular entities may include small molecules, nucleic acids, macromolecules such as proteins or protein complexes, polymers, lipids, including lipid micro spheres, nanotechnology based entities, viruses, or any other molecule or molecular entity either singly or in any combination with one or more other molecules or molecular entities.

[0014] Examples include but are not limited to: small molecular compounds, siRNA, nucleic acids such as expression constructs or other plasmids, peptides and peptide mimics, polypeptides up to and including monomeric, multimeric, homomeric and heteromeric protein complexes, antibodies or any fragment thereof (including Fab, F(ab)2, Fv, scFv, peptibodies, phage display antibodies, and multispecific ligands,) soluble receptors or receptor complexes, or any other synthetic, recombinant, or naturally occurring molecule or complex of molecules that may be delivered to a cell or organism comprising at least one cell in the context of a screening assay.

[0015] The disclosed screening assay assesses the activity of KIBRA with regard to its localization within a cell. The cell may be any prokaryotic or eukaryotic cell, nucleated or enucleated, located in vivo, in vitro, or ex vivo. The cell may be derived from a bacterium, fungi, blue-green alga, plant or animal.

[0016] The cell may be naturally occurring or human made and may be configured in such a way to express a particular genetic target as through stable or transient transfection with a plasmid containing the target. The assay may assess any subcellular localization of

KIBRA including but not limited to the nucleus, cytoplasm, mitochondria, Golgi, lysosome, endoplasmic reticulum, vacuole, synapse, neurate, dendrite, chloroplast, cell wall, or any other subcellular fraction, space, structure, or organelle. The cell may be in vitro, in vivo, or ex vivo when the assay is performed.

[0017] Cells to be used in the assay are transfected with an expression construct

(which may also be known as an expression vector) that includes the KIBRA gene and a reporter gene spliced in such a way that expression of KIBRA results in coexpression of the reporter gene with KIBRA such that KIBRA expression may be visualized. An expression vector may be any nucleic acid, that may be used to introduce a desired gene into a cell such that the mRNA or protein encoded by the gene may be expressed by the cell's transcription and translation systems. The construct may include promoters or enhancer elements that facilitate transcription of the gene. The promoters or enhancers may be tissue specific or continually active. A reporter gene may be any gene that results in the expression of a protein that may be visualized by an instrument or the aided or unaided human eye. Reporter genes allow visualizations by any of a variety of methods.

[0018] Examples include but are not limited to: giving off light directly or as a result of fluorescence emission, rendering or subcellular locations a particular color differentiable from background, allowing the cell to survive in the presence of a material toxic to the cell, or allowing the cell to survive in the absence of a nutrient essential to the cell. Examples of reporter genes include but are not green fluorescent protein (GFP), lucif erase, dsRed, lacZ, X-gal, and chloramphenicol acetyltransferase. The reporter construct is coupled to KIBRA in such a manner that localization of the reporter within the cell indicates that KIBRA has the same localization within the cell. An example is a fusion construct. Confirmation of such a correlation may be assessed the use of proper negative and/or positive controls. [0019] Addition of a molecular entity to a cell encompasses any action that brings the molecular entity into sufficient contact with the cell that the molecular entity, if it is capable of affecting the biological activity to be tested, will do so. The concept of application of the molecular entity to the cell includes adding the molecular entity to the cell culture media, giving the molecular entity to an animal that comprises the cell (orally, intranasally, intraperitoneally, intravenously, or through any other method known in the art) transfecting the cell with a construct comprising a DNA sequence that encodes the molecular entity and relevant expression sequences into the cell and placing the cell under conditions such that the molecular entity is expressed.

[0020] A construct includes any recombinant DNA molecule that comprises a coding sequence that produces a product comprising a protein or a fragment of a protein. Such constructs may include sequences that confer immunity to a drug that may be added to the culture media such that only properly transfected cells are selected. Constructs may also include sequences that cause constitutive or reversible expression of the coding sequence such as promoters, enhancers or silencers, examples of which are well known in the art. Transfection of a construct into a cell may occur through any of a number of methods well known in the art, including electroporation, lipid-based transfection reagents, viral transfection, or any other method now known or yet to be invented that introduces a nucleic acid sequence into a cell for the purposes of expressing one or more specific molecules derived from that nucleic acid.

[0021] Expression encompasses all processes through which material derived from a nucleic acid template may be produced. Expression thus includes RNA transcription, mRNA splicing, protein translation, protein folding, post-translational modification, membrane transport, associations with other molecules, addition of carbohydrate moeties to proteins, phosphorylation, protein complex formation and any other process along a continuum that results in biological material derived from genetic material. Expression also encompasses all processes through which the production of material derived from a nucleic acid template may be actively or passively suppressed. Such processes include all aspects of transcriptional and translational regulation. Examples include heterochromatic silencing, transcription factor inhibition, any form of RNAi silencing, microRNA silencing, alternative splicing, protease digestion, posttranslational modification, and alternative protein folding. [0022] A specific gene such as KIBRA or a reporter gene may be identified by the sequence of a nucleic acid from which it can be derived. Examples of such nucleic acids include mRNA, cDNA, or genomic sequences. Alternatively, a specific gene may be identified by a protein sequence. However, the specific gene is not limited to the products of the exact nucleic acid sequence or protein sequence by which it may be identified. Rather, a specific gene encompasses all sequences and subsequences that express an active or inactive form of the gene.

[0023] Examples of sequences encompassed by a specific target identified by a nucleic acid molecule include point mutations, silent mutations, deletions, frameshift mutations, translocations, alternative splicing derivatives, differentially methylated sequences, differentially modified protein sequences, and any other variation that results in a product that may be identified in combination with a reporter gene. The expression construct may contain truncated, extended, mutated, or otherwise altered forms of the KIBRA gene. Further, the expression may occur in the absence of proteins that prostranslationally modify the KIBRA gene, thus resulting in an alternatively post-translationlaly modified KIBRA protein.

[0024] If addition of a molecular entity to a cell containing the aforementioned

KIBRA expression construct alters the cellular location of KIBRA, as visualized by altered location of the reporter gene and has no effect on a control cell (for example, a control cell that contains the expression construct but was not treated with the molecular entity,) the molecular entity then has an ability to regulate KIBRA activity.

[0025] In some aspects of the invention, the punctate quality of the localization may be quantitated. Though any method of numerically representing the localization of the reporter gene within the cell may be used. One such example is given below (adapted from

Glynn and McAllister, Immunocytochemistry and Quantification of Protein Colocalization in

Cultured Neurons, Nature Protocols I, 1287-1296 (2006). This reference is hereby incorporated by reference in its entirety.)

[0026] The protocol may be performed with a laser scanning microscope with ability to resolve the reporter gene. The protocol may be performed with any software program that will allow manual selection of punctae or adaptive background subtraction (examples include ImagePro from MediaCybernetics or Metamorph ImagingSeries software from

Molecular Devices.

[0027] First, collect fluorescence images. Select the appropriate objective. Set the intensity of the laser as sufficiently low as to avoid photobleaching during imaging. Set the sensitivity (gain) of the detector to obtain the full range of fluorophore signal intensity values. Saturation of signal can be indicated by applying a Hi-Lo look-up-table and should be reached by only a few of the most intense punctae in the image. Collect images at the highest resolution possible (1024 x 1024 pixels at a minimum) to aid quantification.

[0028] To allow density determinations in the quantification stage of analysis, mark each image with a scale bar appropriate for the objective and degree of false zoom used.

Import image files at high color resolution, at Least 8 bits per pixel, 1024 x 1024 pixels per image field. Separate color channels. Convert each color image to grayscale. This reduces visual bias in detecting signal in images of different colors. For example, it is easier for the eye to discern intensity hot spots in a green than in a blue false-colored image. Select neuronal processes for analysis. Select at least three neuronal processes (axons or dendrites) 70-100 um in length along similar distances out from the soma on each of five cells from each of two culture preparations.

[0029] Next, set the size of the region of interest (ROI). Most software programs have a tool that allows the size of the region of interest ROI to be set manually. This can be calibrated with a scale bar. ROIs designating the Location of each punctum and punctae selected for background staining are marked and accumulated into a 'mask' to describe the distribution of each reporter gene.

[0030] Then, manually select the punctae. Punctate regions of fluorescence intensity are defined as focal areas of intensity greater than the average local background fluorescence plus two times the standard deviation. Make a mask of ROIs identifying every punctum on a single gray-scale Image.

[0031] Layer the first mask onto the second gray- scale image (representing a second antibody stain), and add ROIs identifying the location of punctae on the second image not labeled by the initial mask. Layer the mask onto each additional gray- scale image, and complete it with ROIs identifying the location of punctae on each additional image not yet labeled in the mask. Collect intensity values for each point in the master mask on each of the channel images of interest.

[0032] Next, select background and subtract diffuse background staining. Make a new mask of ROIs identifying sites of background fluorescence on each neuronal process used for the selection of puncta within each gray-scale image. Collect intensity values for one or more points in the background masks for each neuronal process for each channel. Calculate the mean local background for each neuronal process and each channel. Add two times the standard deviation to the mean to define the background intensity value. Import intensity values for ROIs selected in each channel into a data analysis program. [0033] Subtract the background value from the intensity value for each neuronal process for each channel separately. All punctae greater than zero after background subtraction are true punctae. Finally, sum the number of ROIs with positive values, and divide by the length of the region analyzed to establish the punctum density. ROIs that include fluorescence from multiple channels indicate colocalized proteins in each channel. [0034] In a further aspect, the present invention provides methods for identification of memory and/or cognition enhancing test compounds, which comprise (1) contacting said test compound with cultured cells and (2) assessing the KIBRA subcellular localization within the cells.

[0035] To screen for compounds able to change KIBRA-distribution one would use a cultured cell line (e.g. PC 12 cells, SH-SY5Y cells, or HEK cells) or primary cells, preferentially neural cells, e.g. hippocampal cells, as the screen addresses neuronal processes. Subcellular distribution of KIBRA with in this method can be assessed with computer aided devices allowing automated image processing in order to allow for higher throughput. Automated image processing allows to transform the subcellular distribution of KIBRA in quantitative parameter, e.g. the ratio of intracellular KIBRA detection versus cell membrane localized KIBRA detection. If necessary, it is possible to facilitate the quantitative analysis by counter- staining of certain subcellular structures such as the cell membrane.

[0036] The quantitative analysis of the subcellular localization of KIBRA allows for statistic comparison of the localization in presence of a test compound to the one in absence of a test compound based on the observation of a number of cells or a number of assays. Whether a subcellular KIBRA distribution measurement is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student' s t-test, Mann- Whitney test etc. Preferred confidence intervals are at least 95 %. The p-values are, preferably, 0.05. [0037] Detection of KIBRA subcellular distribution could be performed by using

KIBRA specific antibodies. Such antibodies can be generated form mouse hybridoma cells, which have been generated e.g. by the immunization of mice using KIBRA- peptides, in order to receive specific monoclonal antibodies. Alternatively, polyclonal antibodies isolated from the serum of animals immunized with KIBRA protein or peptides thereof can be used accordingly. Suitable KIBRA peptides can be derived form the KIBRA protein sequences (e.g. human KIBRA protein (SEQ ID NO: 2), mouse KIBRA protein (SEQ ID NO: 4), or rat KIBRA protein (SEQ ID NO: 6)). [0038] Alternatively to the above described detection of the endogenously expressed KIBRA protein, one can exogenously express KIBRA or suitable KIBRA fusion proteins in the test cells by transfecting the test cells with corresponding expression vectors prior to the contacting of the cells with the test compound. Suitable KIBRA fusion proteins are such proteins which allow for an easy detection of the subcellular localization, e.g. but without any claim of completeness such fusion proteins, where KIBRA is linked to a tag-peptide (e.g. myc-tag) which allows an easy detection by commercially available antibodies, or where KIBRA is linked to a fluorescent protein (e.g. GFP). Within the expression vector the cDNA for KIBRA (e.g. human KIBRA cDNA (SEQ ID NO: 1), mouse KIBRA cDNA (SEQ ID NO: 3), or rat KIBRA cDNA (SEQ ID NO: 5)). or a KIBRA fusion protein is put under the control of a suitable promoter, e.g. SV40 or CMV. To establish the assay method, the subcellular localization of exogenously expressed KIBRA or KIBRA fusion protein should be compared to the one of the endogenous KIBRA in the absence of a test compound in order to avoid artificial subcellular localization due to inadequately high over- expression.

[0039] Test compounds for this assay method can be for example polypeptides, proteins (including chimeric proteins), antibodies, nucleic acids (e.g. siRNA), gene therapy approaches (virus) or small molecules. Preferable for a high throughput approach are small molecules, with a molecular weight of less than 2,000 g. Compounds are generally diluted in salt buffer of physiological concentration (e.g. PBS), hydrophobic compounds, like fat-soluble vitamins, can be diluted in suitable solvents, e.g. 20 % Solutol HS (B ASF, Ludwigshafen, Germany), for efficient application to the cell culture.

[0040] In the view of the above, a method according to the invention that identifies compounds enhancing KIBRA expression or activity is a promising approach for treating and/or preventing cognition impairments, including non-pathological forms like "age related memory loss" (ARML). Cognitively impaired people have difficulty with one or more of the basic functions of their brain, such as perception, memory, concentration and reasoning skills. Common causes of cognitive impairment include Alzheimer's disease and related dementias, stroke, Parkinson's disease, brain injury, brain tumor or HIV-associated dementia.

[0041] Accordingly, one aspect of the present disclosure is the use of compounds as being capable to direct KIBRA protein to a cell membrane localized distribution, for the manufacturing of a pharmaceutical preparation for enhancing memory and cognitive ability, and thereby, treating disease such as Alzheimer's disease, dementia, or "mild cognitive impairment" (MCI) and also nonpathological ARML.

[0042] Also, one aspect of the present disclosure is a method for improving memory or cognitive ability in a subject, the method comprising administering to said subject in need thereof, a therapeutically effective amount of a compound, identified by a method of the invention as being capable to direct KIBRA protein to a cell membrane localized distribution, particularly, whereas said subject suffer from Alzheimer' s disease, dementia, MIC, or non-pathological ARML.

EXAMPLES

[0043] EXAMPLE 1. Differentated PC- 12 cells were co-transfected with KIBRA-

GFP fusion construct and either a control construct [expression the NEO drug resistance gene] or a construct expressing the activated form of PKC-zeta; an upstream kinase that acts on KIBRA. Cells were imaged 36 hours later by direct fluorescent microscopy. The two top panels illustrate the GFP signal in cells without the presence of activated PKC-zeta while the two bottom panels illustrate the distribution pattern in the presence of activated PKC-zeta. Therefore, the disclosed screening assay quantitates the punctate quality of the GFP signal. [0044] EXAMPLE 2. Subcellular localization of KIBRA depends on PKC zeta activation. To access the extent of KIBRA subcellular redistribution in a cell culture assay dependent on PKC zeta activity, PKC zeta was activated by a myristinylation taq. It is generally accepted that such a PKC zeta activation leads to cascade of events resulting in the translocation of the kinase to the cell membrane, which is induced by second messengers or effectors which interact with the regulatory domain of the kinase. As a myristinylation taq tracks PKC zeta to the cell membrane, cell membrane-localized PKC zeta can be regarded as the activated form of the kinase. [0045] COS cells were either co-transfected with KIBRA and PKC zeta- myristinylated or KIBRA construct and empty vector control constructs. After 48 hours in culture, cells were immuno stained with KIBRA antibody, visualized with Alexa 488, and PKC zeta antibody labeled by Cy3. Alternatively KIBRA subcellular localizatiuion has been visualized with KIBRA-EGFP fusion construct under the control of the constitutive cytomegalovirus (CMV) promoter. Fluorescent microscopy analysis of cells expressing KIBRA-EGFP alone reveals a punctuated staining (Fig 2A). In contrast, analysis cells expressing KIBRA-EGFP in combination with PKC zeta- myristinylated revealed redistriburion of KIBRA from a punctuated staining to a membrane localized staining (Fig. 2B). These data indicate, that KIBRA subcellular localization is influenced by the pro-cognitive active PKC zeta. [0046] There is a need for reliable, efficient and non-laborious in vitro or cell based methods for the identification of new memory modulating compounds and means which can be performed in a medium to high through put setup. These screening methods need easy to measure and robust readouts to allow multiple testing of compounds or treatments. The embodiments characterized in the claims and herein provide such methods based on the linkage between KIBRA and memory performance. Accordingly, the present disclosure provides for in vitro or cell-based methods for the identification of compounds and means capable of modulating memory performance or cognition based on the assessment of KIBRA' s cellular and subcellular localization. [0047] Various embodiments of the invention are described above in the Detailed

Description. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and/or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well. Unless specifically noted, it is the intention of the inventor that the words and phrases in the specification and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s).

[0048] The foregoing description of a preferred embodiment and best mode of the invention known to the applicants at this time of filing the application has been presented and is intended for the purposes of illustration and description. It is not intended to be exhaustive or limit the invention to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiment was chosen and described in order to best explain the principles of the invention and its practical application and to enable others skilled in the art to best utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims.