MXPA00001318A - Transgenic animals expressing human p25 - Google Patents

Abstract

The invention provides transgenic, non-human animals and transgenic non-human mammalian cells harboring a transgene encoding a p25 (activator of the protein kinase cdk 5) polypeptide. The two neuropathological lesions associated with Alzheimer's disease (AD) are amyloid plaques and neurofibrillary tangles (NFTs), composed predominantly of amyloid beta peptides and hyperphosphorylated tau, respectively. While animal models for plaque formation exist, there is no animal model that recapitulates the formation of NFTs. This invention provides transgenic mice that overexpress human p25, an activator of cdk5, resulting in tau that is hyperphosphorylated at AD-relevant epitopes. Deposition of tau is detected in the amygdala, thalamus and cortex. Increased phosphorylated neurofilament, silver-positive neurons and neuronal death are also observed in these regions. We conclude that the overexpression of p25, an activator of cdk5, is sufficient to produce hyperphosphorylation of tau and neuronal death. The p25 transgenic mouse represents the first model for tau pathology in AD.

Description

TRANSGENIC ANIMALS EXPRESSING HUMAN P25 POLYPEPTIDE DESCRIPTIVE MEMORY The present invention provides transgenic non-human animals and non-human mammalian transgenic cells that harbor a transgene encoding a p25 polypeptide, an activator of the cdkd protein kinase. The invention also provides non-human animals and cells comprising a transgene encoding a p25 polypeptide, and further comprising the over-expression of functional p25, the p25 transgene and blunting constructs used to produce said transgenic cells and animals, transgenes encoding human p25 polypeptide sequences, and methods of using the tansgenic animals in the selection of pharmaceutical compounds, as well as animals for commercial research to model neurodegenerative diseases such as Alzheimer's disease and p25 / cdk5 biochemistry in vivo. Throughout the specification, numerous publications are cited. These publications are hereby incorporated by reference in their entirety. A complete list of publications appears later in the specification. Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by loss of cognitive function. The primary neuropathological lesions in AD are amyloid plaques and neurofibrillary tangles (NFTs). The amyloid plaques are formed mainly of amyloid beta peptides (Ab), varying in length from 39-42 amino acids, which are derived from the amyloid precursor protein (APP) (reviewed in 1). The NFTs are formed of the microtubule-binding protein tau that is hyperphosphorylated in epitopes that exist in a predominantly non-phosphorylated state in disease-free brain (2-4). The respective functions that these lesions play in the neuronal loss and dementia observed in patients with AD continue to be a controversy. The precise mechanism of formation of NFTs is not clear, but research from many laboratories suggests that tau hyperphosphorylation can be an important event. The paired helical filament (PHF) is the fundamental unit of the NFT. Tau hyperphosphorylation in serine or threonine residues followed by proline (SF3 or TP), epitopes that are concentrated in the amino and carboxy termini of tau, results in loss of affinity for microtubules, and a presumed concomitant increase in concentration of cytoplasmic tau (5-8). However, the phosphorylation of tau in serine 262, which is not followed by proline, can also reduce the affinity of tau for microtubules (9). In vitro experiments with purified tau show that in the presence of endogenous cations (for example, messenger RNA, proteoglycan-heparin sulfate), tau is polymerized to form structures indistinguishable from the PHF observed in the brain with AD (10,11). . The formation of PHF dependent on cations in vitro is independent of the tau phosphorylation state, suggesting that the key permissive event at the onset of PHF formation may be an increase in the cytoplasmic concentration of tau. In support of this hypothesis, recent evidence (12-14) indicates that mutations in the tau gene associated with susceptibility to a form of inherited dementia, called frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), reduce affinity of tau for microtubules (15). Therefore, these mutations, like hyperphosphorylation, can result in an increase in cytoplasmic tau concentrations. In the presence of endogenous cations, it is assumed that this increase allows the formation of PHF followed by NFT assembly and, finally, neuronal death. As with other phosphoproteins, the phosphorylation state of tau is the sum of the activity of protein kinase and protein phosphatase. In this way, tau hyperphosphorylation in AD can be due to an increase in kinase activity, or a decrease in phosphatase activity. Although many protein kinases phosphorylate tau in relevant epitopes in AD in vitro (reviewed in 16, 17), only two have been co-purified with mammalian brain microtubules, GSK3b and cdkd (18). As a matter of knowledge, only these two kinases will phosphorylate tau when they are transfected heterologously into mammalian cells (19, 24). We chose to focus attention on cdk5 against GSK3b, since the latter plays a role in energy metabolism, and is actively expressed in all cells, whereas cdkd is only active in neurons (see above). The cdk5 kinase is a member of the family of cyclin-dependent protein kinases, and is expressed in almost all cells (reviewed in 25, 26). Unlike other members of the cdk family, there is no known cyclin that activates cdkd. Rather, the positive allosteric regulators of cdkd are p35 (27), amino terminal proteolytic fragments of p3d, eg, p2d, p23 or p21 (28, 29) and p39 (30). These proteins share minimal homology of amino acid sequences with cyclins (27-29), but the elaboration of computer models and biochemical experiments suggest that the activation mechanism of cdkd by p2d / 3d may be similar to that of activation of cdk2 by cyclin A (31-33). the p2d / 3d protein is expressed predominantly in neurons, implying that most of the activity of cdkd is concentrated in neuronal structures (27,28). The p3d protein has a relatively short half-life within cells, and is rapidly ubiquitinated, suggesting tight regulation of cdkd activity in neurons (34). The cdkd kinase plays an important role in neuronal development, as evidenced by the abnormal corticogenesis and perinatal lethality of mice that have blocked the expression of cdkd (3d), and perturbations in neuronal migration and early death in mice with blocked expression of p3d (36). In rodent development studies, the maximum catalytic activity of cdkd occurs in E11 or 12, which further supports the cdkd function in neurogenesis (37,38). In addition, in cultured primary neurons, cdkd / p3d is located towards the growth cones, suggesting a function in the new growth of neurites (39). Recently, evidence has emerged of signaling pathways that can modulate the activity of cdkd. For example, it has been shown that cdkd / p3d interacts with Rae, and that it modulates the activity of PAK (40), and that the new growth of cerebellar neurons increased by laminin is interrupted by the suppression of p3d expression (41). A few substrates have been identified for cdkd, 0 and are consistent with the putative function in new neurite growth and plasma membrane dynamics. These include cytoskeletal proteins such as tau (42-4d) and neurofilaments (46-48), synaptic vesicle proteins such as synapsin and Munc-18 (49-dO) and the retinoblastoma protein (d1). However, a clear picture of a signal transduction pathway regulating the activity of cdkd / p3d, nor the function of cdkd / p3d in the mature brain has not been elucidated. There is also no clear picture of the protein kinases responsible for the hyperphosphorylation of tau in AD. However, evidence is accumulating that cdkd can perform a pathological function. For example, in vitro studies, cdkd will phosphorylate up to eight different tau epitopes, including those associated with AD, and which are known to decrease tau affinity for microtubules (42-4d). In addition, the heterologous cotransfection of cdkd / p2d with tau in mammalian cells also results in the phosphorylation of several of these epitopes (24). Finally, immunohistochemical evidence suggests that cdkd is close to the NFTs in the brain with AD (d2-d3). The development of experimental models of Alzheimer's disease that can be used to better define the underlying biochemical events involved in the pathogenesis of AD would be highly desirable. These models could be used, in an application, to select agents that alter the degenerative course of the AD. For example, a model AD system could be used to select environmental factors that either induce or accelerate the pathogenesis of AD. Alternatively, an experimental model could be used to select agents that inhibit, prevent or reverse the progression of AD. Such models could be used to develop pharmaceutical compounds that are effective in preventing, counteracting or reversing AD. Only human and non-human primates do not develop some of the pathological characteristics of AD. The cost and difficulty of using primates, and the time it takes to develop the pathology of AD, makes the exhaustive research on such animals prohibitive. Rodents do not develop AD, even at an extreme age. Despite several reports that 0 indicate that certain treatments result in the hyperphosphorylation of tau and / or neuronal death associated with tau phosphorylation, there is a need in the art for non-human transgenic animals that can cause tau hyperphosphorylation and death. neuronal associated.

Based on the foregoing, it is clear that there is a need for non-human cells and non-human animals that can produce hyperphosphorylation of tau and death of neuronal cells. Thus, an object of the present invention is to provide methods and compositions for transferring transgenes and homologous recombination constructs in mammalian cells, especially in embryonic stem cells. It is also an object of the present invention to provide transgenic non-human cells and transgenic non-human animals harboring transgenes, resulting in increased expression of p2d, an activator of 0 cdkd. Of more interest for the present invention is the application of said transgenic animals as live systems to select test compounds with the ability to inhibit or prevent the production of hyperphosphorylated tau and associated neuronal death. It is desirable to provide methods and systems for selecting test compounds for their ability to inhibit or prevent phosphorylation of tau and associated neuronal death. In particular, it will be convenient to base such methods and systems on the inhibition of cdkd / p2d, wherein the test compound blocks the tau phosphorylation mediated by cdkd / p2d, where the test compound also blocks neuronal death. Such transgenic methods and animals should provide a rapid, economical and suitable way to select large numbers of test compounds. Human p2d was expressed excessively in the brains of mice to determine whether an increase in cdkd activity would result in hyperphosphorylation of tau in relevant epitopes in AD, and whether this hyperphosphorylation would lead to neuronal death. In the brains of transgenic mice in p2d, both tau and neurofilaments are hyperphosphorylated, and many silver-positive neurons with tangle-like inclusions are present. Silver-positive neurons suggest ongoing neuronal death. These results demonstrate that the excessive expression of a cdkd activator is sufficient to produce phosphorylation of tau and neurofilaments and neurons positive to silver, which are very similar to those observed in AD. The transgenic mouse in p2d can function as a model for neurofibrillary pathology and neuronal death observed in AD.

BRIEF DESCRIPTION OF THE INVENTION In one embodiment, the present invention is directed to recombinant DNA comprising a rat neuron-specific enolase promoter operably linked to a sequence encoding p2d of the sequence encoding the human cdkd gene. In a preferred embodiment, the present invention is directed to recombinant DNA, wherein said sequence encoding said p2d fragment is genomic DNA. In another preferred embodiment, the present invention is directed to recombinant DNA, wherein said sequence encoding said p2d fragment is cDNA. In still another preferred embodiment, the present invention is directed to recombinant DNA, wherein said sequence is that of SEQ ID NO: 4. In yet another embodiment, the present invention is directed to a vector comprising recombinant DNA in accordance with present invention. In another embodiment, the present invention is directed to eukaryotic cell lines comprising recombinant DNA in accordance with the present invention. In another embodiment, the present invention is directed to a transgenic non-human animal, or progeny thereof, whose germ cells and somatic cells express recombinant DNA according to the present invention. In a preferred embodiment, the present invention is directed to a transgenic non-human animal, or progeny thereof, which is a mouse. In a further embodiment, the present invention is directed to a method for treating an animal suffering from a disease characterized by the expression of a p2d fragment of a human cdkd gene., which 0 comprises administering a therapeutically effective amount of an inhibitor of said p2d fragment. In another embodiment, the present invention is directed to a method for determining the ability of a compound to inhibit the expression of a p25 fragment of a human cdkd gene, which comprises the steps of: a. creating a transgenic non-human animal by stably incorporating into the embryonic stem cells of said animal, the recombinant DNA of claim 1; b. developing said embryonic stem cells in a mature transgenic non-human animal; c. administering to said transgenic non-human animal the compound of interest; and 0 d. measuring the inhibition of said fragment of p2d by said compound. In yet another embodiment, the present invention is directed to a method for generating data for determining the ability of a compound to inhibit the expression of a p2d fragment of a human cdkd gene, d which comprises the steps of: a. creating a transgenic non-human animal by stably incorporating into the embryonic stem cells of said animal, the recombinant DNA of claim 1; b. developing said embryonic stem cells in a mature transgenic human or non-human animal; c. administering to said transgenic non-human animal the compound of interest; d. measuring the inhibition of said fragment of p2d by said compound; and e. using the data derived from said inhibition to synthesize compounds capable of inhibiting said p2d fragment. Figure 1A shows Western blots of the amygdala (1), thalamus d (2) and cortex (3) of 3 wild type mice that were treated with probe with an antibody specific for p2d and p3d / 39 (generous gift of L. -H. Tsai, Harvard Medical School Cambridge, MA). Figure 1B shows Western blots of the amygdala (1), thalamus (2) and cortex (3) of 3 transgenic mice in p2d which were treated 0 with probe with the same antibody as in Figure 1A. The expression of the p2d transgene is apparent in the transgenic mice, but not in the wild-type mice. Figure 2 shows the brain of a 4-month transgenic mouse immunopositive for AT-8 (a commercially available antibody, which specifically recognizes phosphoserine 202 / 20d of tau) in the rostral portion of the amygdala. The bodies of neuronal cells with accompanying axons (arrows) are positive. Several dark brown positive cells are observed in this field. Figure 3 shows the brain of a 4 0 month old wild type mouse minimally positive for AT-8 in the rostral portion of the amygdala. A cell body exhibits non-specific positivity (arrow) due to secondary antibody. Figure 4 shows the brain of a transgenic mouse of 4 months demonstrating PHF-13 (an antibody that recognizes phosphoserine 396/404 tau; (gift from Dr. V. Lee, U. of Pennsylvania), showing immunopositivity in the rostral portion of the amygdala The body of the neuronal cell with a thickened axon (arrow) is positive A positive neuronal cell body (arrowhead) with swollen / atrophied axon is also possible Figure 5 shows the brain of a 4-month wild-type mouse minimally positive for PHF-13 in the rostral portion of the amygdala Cell bodies exhibit non-specific positivity (arrows) due to the secondary antibody Figure 6 shows the brain of a 4-month transgenic immunopositive mouse total tau (an antibody that recognizes total tau, commercially available from Accurate Chemical and Scientific Corp Westbury, N. Y.) in several cell bodies of the rostral amygdala (examples marked by arrows). Figure 7 shows the brain of a 4 month immunopositive wild type mouse for tau in axons (arrowheads) of the rostral amygdala. Figure 8 shows the brain of a transgenic mouse of 4 months that was reacted with SMI-34 (a commercially available antibody that recognizes the H protein of phosphorylated neurofilaments). Positive cells (arrows) are observed in the rostral amygdala. Figure 9 shows minimal axonal positivity of SMI-34 in the rostral amygdala of the brain of a 4-month wild-type mouse. Figure 10 shows silver-positive cell bodies (an indication of disintegrated cytoskeleton and neuronal cell death) (arrows) in the rostral amygdala of the brain of a 4-month transgenic mouse, d Figure 11 shows minimal positivity of axons to silver in the rostral amygdala of a 4-month wild-type mouse. Figure 12 shows several dilated axons (arrows) that show positivity to silver in the spinal cord of a transgenic mouse of d months. Figure 13 shows several dilated axons (arrows) that show positivity to SMI-34 in the spinal cord of a transgenic mouse of d months. Figure 14 shows that normal axons in the spinal cord of a 6-month wild-type mouse are negative for SMI-34. In general, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, chemistry and nucleic acid hybridization, biochemistry, histology and immunocytochemistry described below, are well known and are commonly described in the art. Standard techniques are used for recombinant nucleic acid methods, polynucleotide synthesis, cell culture, transgene incorporation, Western blotting, immunocytochemistry, and histological techniques such as silver staining. The techniques and procedures are generally carried out in accordance with conventional methods in the art, and several general references are provided throughout this specification. The methods of the present are well known in the art and are provided for the convenience of the reader. All the information contained herein is incorporated herein by reference, In accordance with the above objects, in one aspect of the invention non-human animals are provided which house at least one copy of a transgene comprising a polynucleotide sequence. which encodes a heterologous protein operably linked to regulatory elements that are capable of expressing the heterologous protein in the transgenic non-human or animal. Said heterologous polypeptide is a truncated portion of the cyclin dependent p3d kinase regulatory protein p3d (cdkd) (27, 28). The truncated heterologous polypeptide has the first 98 N-terminal amino acids removed, and has a molecular weight of approximately 2d, 000 daltons. This novel polypeptide will be referred to as p2d. Typically, the transgenic non-human animal is a mouse, and the heterologous gene is the human p2d sequence. Transgenes are typically cDNA sequences that have been operably linked to regulatory sequences that act at the cis-position that direct expression in the transgenic host mammal in a cell-specific manner, typically 0 in neurons. Typically, the transgene will be incorporated into the chromosomes of the host in a non-directed random manner. The invention further provides that the non-human transgenic animal hosts at least one copy of the transgene of the p2d heterologous sequence or vector-driven gene of the invention integrated homologously or non-homologously at the position of the chromosome, expressing the truncated p25 polypeptide. Transgenic animals are typically produced by introducing a transgene by microinjection into pronuclei of single-cell embryos or a vector into the host by electroporation, lipofection or viral transfection of embryonic stem cells (ES). Transgenic animals expressing the p2d polypeptide are suitable for use as disease models and selection of potential therapeutic compounds including polynucleotides, proteins and small molecules. The invention also provides 0 human or non-human cell lines that will be derived by transformation of established cell lines or primary cell lines established directly from the transgenic non-human animal, eg, neurons. It is obvious that the transgenic non-human animal can have additional genetic modifications by transgenic processes or d through traditional pairings with other transgenic animals or that occur naturally to produce novel animals that function as alternative disease models, drug selectors or other applications. This includes the inactivation of the endogenous murine p3d gene through gene mapping in ES cells and resulting murine p3d deficient mice, which are the preferred host for expression of the human heterologous p2d polypeptide. Such heterologous transgenes can be integrated into non-homologous chromosomal sites in the transgenic animal, typically derived from pronuclear injection or can be integrated by directing the gene in ES cells by methods to inactivate the murine p3d gene and add linked sequences to direct expression. of the p2d sequences of heterologous human. In general, the invention encompasses methods for the generation and characterization of transgenic animals expressing human p2d polypeptide, a proteolytic fragment of p3d, both are allosteric activators of cyclin dependent kinase 5 (cdkd). The transgenic animals express this human protein in the presence of the endogenous homologue. The techniques and procedures are carried out according to established protocols that are generally considered as routine methods in the art and references are provided within this specification. When the cdkd protein kinase is associated with an allosteric activator, for example, it is reported in various publications that p3d or p25 phosphorylate tau in vitro and in whole cells (see background of the invention). Thus, the transgenic mice thus produced establish the role of p2d of human in the formation of hyperphosphorylated tau under neurodegenerative conditions including Alzheimer's disease, Parkinson's disease, amylolateral sclerosis, Hungtingon's disease, shock, traumatic brain damage, Pick's disease, neuroaxonal dystrophy, multiple sclerosis, disease of 0 motor neurons and cerebellar spine degeneration, and other neurodegenerative diseases. It is evident that the preparation of other animals expressing human p2d protein is easily achieved among those that include rats, hamsters, guinea pigs and rabbits. Transgenic animals expressing human p25 protein can be monitored to verify the level of tau expression and phosphorylation. It will be appreciated that under different conditions, the level of expression and degree of phosphorylation of tau is inhibited in said animal model. In particular, the selection of therapeutic agents that inhibit the activity of human p2d protein is greatly facilitated in animal models. It is evident that the development of cell lines of the affected transgenic animals (for example, neurons) will improve the development to which the biochemical and pharmacological analysis of the human p2d protein can be evaluated. 0 Particularly preferred animal models for excessive expression of p2d are transgenic animals expressing p2d as described above. Said transgenic animals, particularly the transgenic mice according to the present invention, produce large amounts of p2d, hyperphosphorylated tau and neuronal death that can be detected according to the methods of the present invention. In accordance with the present invention in particular, Excessive expression of p2d will be equal to or greater than the expression of endogenous p2d in said animals. In addition, this level of excessive expression of p2d results in hyperphosphorylation of tau that is minimal in animals of wild type. With such high p2d levels, monitoring of tau hyperphosphorylation and neuronal death is greatly facilitated. In particular, the selection of compounds and other therapies to inhibit tau phosphorylation is unduly simplified in animals expressing p2d excessively in accordance with this invention. The agents are administered to test animals, such as test mice, which are transgenic and which excessively express p2d. The following describes the particular techniques for producing transgenic mice that express excessively p25. It will be considerable that the preparation of other transgenic animals overexpressing p2d is easily achieved, including rats, hamsters, guinea pigs, rabbits and the like. In light of this description, the effect of the test compounds on the tau hyperphosphorylation in the test animals in various specimens of said animals can be measured. The effect of the test agents on tau hyperphosphorylation can be measured in various specimens of these test animals. In all cases, it will be necessary to obtain a control value that is characteristic of the level of phosphorylation of tau and neuronal death in the test animal in the absence of the compound (s). In cases where the animal is slaughtered, it will be necessary to base such control values on an average or a typical value of other test animals that have been transgenically modified to express excessively p2d but have not received the administration of any test compound or of any other substance which is expected to affect the level of tau phosphorylation or neuronal death. Once that level of control is determined, test compounds can be administered to additional test animals, where the deviation from the average control value indicates that the test compound had an effect on tau phosphorylation or neuronal death in the animal. Test substances that are considered positive, ie, that are considered beneficial in the treatment of AD or other neurodegenerative diseases, will be those that can reduce the level of d phosphorylation of tau or neuronal death preferably at least 20% and most preferably 80% In addition, there may be formation of straight or helical coiled filaments in the transgenic animals that express excessively p2d and exhibit tau hyperphosphorylation. In these cases, the test compounds can be administered to the test animals and the reduction in filament formation can be monitored as a result of exposure to the compound. In addition, there may be behavioral alterations in transgenic animals that excessively express p2d and exhibit hyperphosphorylation of tau. In these cases, it will be necessary to obtain a control value of live animals that develop a behavioral task d (for example, the measurement of locomotor activity) in the test animal in the absence of the compound (s). Such control will also be determined in non-transgenic wild-type mice. The difference between the wild-type and the transgenic mice will be useful as the measure of the result of the effects of the compounds. Once the control levels are determined, 0 the test compounds can be administered to additional test animals, where the reduction in or reversal of the difference between the wild-type and the transgenic mice indicates that the test compound has a effect on the behavioral test that has been measured. Test substances that are considered positive, that is, considered to be beneficial in the treatment of AD or other neurodegenerative diseases, preferably those that are capable of reversing or, substantially reversing, or favorably modifying the behavioral abnormality in the transgenic animal to the level found in the wild-type mouse. The test agents will be defined as any small molecule, protein, polysaccharide, deoxy or ribonucleotide, or any combination thereof which, when added to the cell culture or to the animal will not interfere in the opposite way with the viability of the cell or of the animal Agents that alter the level of human p2d expression and tau phosphorylation will be considered as candidates for further evaluation as potential therapeutic agents. The test compound will typically be administered to the transgenic animals at a dose of d 1 ng / kga1 OOmg / kg generally from 10μg / kg to 32mg / kg. Test compounds that are capable of inhibiting tau phosphorylation will be considered as candidates to further determine the ability to block tau phosphorylation in animals and in humans. Inhibition of tau phosphorylation indicates that the cdkd / p2d activity has been at least partially blocked, reducing the amount of cdkd / p2d available to phosphorylate tau. The present invention further consists of pharmaceutical compositions that incorporate a compound selected by the method described above and include a pharmaceutically acceptable carrier. Said pharmaceutical compositions should contain a therapeutic or prophylactic amount of at least one compound identified by the method of the present invention. The pharmaceutically acceptable carrier can be any compatible, non-toxic substance suitable for delivering the compound or compounds to a target host. Sterile water, alcohol, fats, waxes and inert solids can be used as the mentioned vehicle. Pharmaceutically acceptable adjuvants, pH regulating agents, dispersing agents and the like can also be incorporated into the pharmaceutical compositions. The preparation of pharmaceutical compositions incorporating active agents is suitably described in medical and scientific publications. See, for example, Remigton's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. 16th Ed., 1982, the description of which is incorporated herein by reference. The pharmaceutical compositions that were recently described are suitable for systemic administration to the host, including both parenteral, topical, and oral administration. The pharmaceutical compositions can be administered parenterally, i.e., subcutaneously, intramuscularly or intravenously. Therefore, the present invention provides compositions for administration to a host where the compositions consist of a pharmaceutically acceptable solution of the identified compound in an acceptable carrier, as described above.

Uses for research and commercial selection Non-human animals comprising transgenes encoding p2d can be used commercially to select agents that have the effect of preventing or reducing the phosphorylation d of tau and neuronal death. Such agents can be developed as pharmaceutical agents to treat hyperphosphorylation of tau and AD among other neurodegenerative diseases. For example, knockout mice of Donehower pd3 and others (d4) have been widely accepted as commercial products for screening carcinogens and the like. The transgenic animals of the present invention exhibit abnormal tau phosphorylation and can be used for selection of pharmaceutical agents and as models for neurodegenerative diseases and the biochemistry of cdkd / p2d and tau. Said animals have various uses including, but not limiting the use to identify compounds that affect the hyperphosphorylation of tau, in a variation, the agents are identified as candidate pharmaceutical agents. Transgenic animals can be used to develop agents that modulate the expression of cdkd or p2d and / or its stability; said agents may be useful as therapeutic agents to treat neurodegenerative diseases. Mice expressing excessively 0 p2d of this invention may also be useful as disease models for investigating pathologies related to tau (e.g. AD, Pick's disease, Parkinson's disease, temporal frontal lobe dementia associated with chromosome 17, shock, traumatic brain injury, moderate cognitive dysfunction, and the like). Such transgenic animals can be commercialized for researchers, among other uses. As the invention has already been described in general terms, reference will be made below to the specific examples. It is necessary to understand that these examples should not limit the present invention, the scope of which will be determined by means of the appended claims. Plasmid pNSRE-p2d (rat neuron-specific enolase promoter, p2d transgene from human) contains DNA from 4 0 different sources: rat neuron-specific enolase promoter; the human cDNA for the p2d catalytic fragment of the p3d protein; the SV40 polyadenylation signal sequence and a commercially available plasmid vector. The transgene is isolated by digestion of pNSE-p2d by means of the restriction enzyme of BamHL This digestion by restriction releases the transgene NSE-p2d (3212-bp) of the plasmid cloning vector pSP72 (2462bp) (Promega, Madison Wl ). The rat neuron specific enolase promoter (NSE) sequence is 1826bp (SEQ ID NO: 1) and has been shown to efficiently express the sequences encoding DNA in sequences in neurons of transgenic (dd) mice. 0 The coding region for the human p2d sequence is 633bp. (SEQ ID NO: 2). The 3 'untranslated primer and the SV40 polyadenylation sequence are 688bp (SEQ ID NO: 3). The micro-injected NSE-p2d transgene is 3213bp (SEQ ID NO: 4). Enzymatic reactions of the recombinant DNA including ligations, restriction endonuclease digestions, DNA synthesis reactions, single chain completion reactions and developed bacterial transformations are already established procedures as described by Sambrook, J., Fritsch, E.F. and Maniatis, T. d Molecular Cloning. A Laboratory Manual. 2pd edition, Cold Spring Harbor Press, New York, 1989. The SV40 polyadenylation sequence (SEQ ID NO: 3) was cloned into the commercially available pSP72 plasmid (SEQ ID NO: d) at the Xbal and BstXL sites. This plasmid, designated as pSP-SVpa, it served 0 as the basic vector for the construction of the transgene plasmid NSE-p2d. The cDNA for the human p2d catalytic fragment was derived using polymerase chain reaction (PCR). The full-length human cDNA of p3d was used as a template for the amplification of the p3d insert. The p3d template DNA was received from Dr. David Auperin of Pfizer; the human p3d sequence was cloned into the EcoR1 site of pFastBad, from the baculoviral expression vector (Gibco-BRL, Gaithersburg, MD). PCR primers were designed to have Notl 1 restriction sites outside of the coding sequence to facilitate the construction of transgene. The forward primer also introduced a new ATG 0 start codon adjacent to the GC (Ala codon at position 99 of the human p3d sequence). The primers (subjected to the 3 'orientation) were p2d forward: GCGGCCGCATGGCCCAGCCCCCACCGGCCCAGCCGCCTGCA (SEQ ID NO: 6) and reverse p2d: CTCCTCCTAGGCCTGGAATCGGTGAGGCGGCCGC (SEQ ID NO: 7). The ATG is introduced into the start codon, the TGA is the stop codon and the GCGGCCGC are the Notl restriction sites added to the PCR primers. The resulting PCR product is 648 bp containing the total 633 bp p2d human cDNA sequence with genetically engineered ATG start site (SEQ ID NO: 2) and an additional 15 bp for the addition of Notl cloning sites. The 648 bp fragment was subcloned into the vector pCR2.1 (Invitrogen, Carlsbad, CA) for verification of the pre-use sequence as an insert in the final transgene. The confirmed 648 bp Notl fragment was ligated into the NotI restriction site of pSP-SVpA, yielding the pSP-SVpA: p25 plasmid. The rat neuron-specific enolase (NSE) promoter was isolated from the pNSE-lacZ plasmid obtained from Dr. J.G. Sutcliffe of the Research Institute of Scripps Clinic (LaJolla, CA). The rat NSE promoter sequence (SEQ ID NO: 1) was isolated by EcoR1 and the Hind III restriction digestion of the pNSE-lacZ plasmid. The 1.8 kb fragment was extracted by agarose gel electrophoresis and purified. The d 'single chain extensions generated by the restriction enzymes were filled by the Klenow reaction to produce the rat NSE promoter fragment with shaved double stranded ends. The shaved-end promoter fragment of rat 1826 bp NSE was ligated at the shaved end to the Sma1 restriction site of pSP-SVpA: p2d. The correct orientation of the rat NSE promoter in the pSP-SVpA: p2d was verified by the double restriction digests of BamH1 and Xhol. The plasmids with the correct orientation were designated pNSE-p2d. The total sequence of the 3212 bp NSE-p2d transgene (SEQ ID NO: 4) was analyzed and verified by automatic sequencing on the ABI 373 sequencer (Foster City, CA) using the standard dye terminator chemistry protocol delineated by ABI . All plasmids were cultured in DHdalfa bacterial cells (Gibco BRL Gaithersburg MD).

Production of transgenic mice expressing excessively human p2d The 3212 bp NSE-p2d transgene DNA fragment was extracted from the plasmid pNSE-p2d by restriction endonuclease reaction BamHL The 3.2 kbp fragment was isolated by electroelution (dOV, 3 hours ) after electrophoresis on 1% agarose gel (FMC Bioproducts, Rockland ME). The fragment was further purified on a Schleicher and Schuell (Keene, NH) Elutip-d column following the protocols established by the manufacturer for the purification of DNA prior to the microinjection of murine embryos. The production of transgenic mice by pronuclear microinjection was carried out by published procedures as outlined in Hogan, B. et al Manipulating the Mouse Embryo: A Laboratory Manual Second Edition, Cold Spring Harbor Laboratories, New York, 1994. Embryos from pronuclear stage of the F1 female mice of the FVB / N strain (Charles River Labs, Wilmington, MA) were obtained after superovulation with international units (IU) of pregnant mare serum follicle stimulation hormone (Sigma St. Louis, Mo.) and 2.5 IU of chorionic gonadotropin (Sigma). The actual microinjection procedure was carried out as described by Wagner, T. et al (56), except that the embryos were immediately transferred to the female ds pseudopregnant CD-1 recipients (Charles River Laboratories, Wilmington, MA) for the final development of the embryos. Mice resulting from the reimplantation events were tested for the presence of the NSE-p2d transgene by PCR analysis of the genomic DNA isolated from the tail biopsies at the age of 3 weeks. Mice that demonstrated 0 to be positive for the presence of the NSE-p2d transgene were coupled to the wild-type FVB / N mice (Charles River Laboratories, Wilmington, MA) of the opposite sex. The progeny of said couplings was tested for germline transmission by PCR analysis or Southern blot analysis of genomic DNA isolated from tail biopsies at the age of 3 weeks. The transgenic lines were produced from the 7 founding transgenic mice and were maintained by crossing the wild-type FVB / N mice and the PCR genotype in the presence of the NSE-p2d transgene. The experiments described below were done in mice 0 heterozygous for the inserted transgene. Said mice were derived by cross-breeding mice positive for the transgene inserted with wild-type FVB / N animals and selecting descendants by PCR in the presence of the NSE-p2d transgene sequences.

Detection of Excess Expression of the Western Blot p2d Protein All the brains of transgenic (Tg) and wild type (wt) mice from 1 to 4 months were removed and frozen in liquid nitrogen. The tonsils, thalamus and cortex were dissected and homogenized in 1 ml of pH lysis buffer (as described in 37). The mixtures were boiled for 10 minutes and then centrifuged at 13,000 rpm. The protein concentrations of the resulting supernatants were determined by the Pierce BCA method (0 microprotein BCA assay, catalog # 2322d, Pierce, Rockford ILL). Ten micrograms of each sample were subjected to electrophoresis in a SDS polyacrylamide gel (d7) and transferred to the ProBlott protein paper for western blot analysis [western blotting protocols, ECL detection, Amersham Life Science Arlington Heights, ILL ( 199d)]. The non-specific sites were blocked by incubation of the blots in d% of skim milk in tris-regulated saline at their pH (20 mM tris base, 127 mM NACI, 3.8 mM HCl, pH 7.6, Sigma) included 0.1% tween-20 (TBS-T). The primary antibodies were diluted in d% of skim milk / TBS-T. The blots were placed in the diluted antibody solutions 0 and rotated for 1 hour at 23 ° C. The western blots were then washed for 30 minutes in TBS-T. The secondary horseradish peroxidase bound to the antibodies was diluted in d% skim milk / TBS-T. The blots were incubated with the secondary antibody solution and rotated for 4d minutes at 23 ° C. The blots were then washed for 30 minutes in TBS-T. Equal volumes of ECL development solutions from Amersham A and B were mixed. The western blots were incubated for 1 minute in the development solution. The blots were covered with a Sarán ® cover and then exposed to an image film (Kodak Rochester N.Y. X-OMAT AR, catalog # 165 1454). The western blots of the tonsils (1), the thalamus (2) and the cortex (3) of the three mice were tested with an antibody specific for p25 and p35-39 (courtesy of L.-H. Tsai, Harvard Medical School , figure 1A). Although the detection of p3d / 39 is apparent, no p2d was detected in the wild-type mice. When the same analysis was carried out in the transgenic mice (FIG. 1B), the robust expression of p2d was detected, in addition to the constitutive expression of p3d. These results confirm that the expression of the transgene is robust in the p2d transgenic animals. d Silver staining and immunohistochemistry of SMI 34 The tissues harvested from mice with programmed sacrifice were fixed in situ by perfusion with 10% neutral formalin regulated at its pH or 4% paraformaldehyde, while the tissues harvested from the 0 mice were They found dead when the immersion was fixed in formalin. After fixation, the sectioned tissues were dehydrated through graduated alcohols and embedded in paraffin. The standard cross sections of the brain were identified by the analogy of the figures in The Mouse Brain in Stereotaxic Coordinators (Franklin and Paxinos, 1997). Paraffin sections (8 μm thick) were stained with Bielschowsky silver strain and, immunohistochemically, with an antibody directed against phosphorylated neurofilament (SM! 34, commercially available from Sternberger Monoclonals, Inc. Lutherville, MD).

Immunohistochemistry For immunohistochemistry studies, the transgenic and wild-type mice were deeply anesthetized with sodium pentobarbital (60 mg / kg, ip) and perfused through the ascending aorta with 20 ml of 0.1 M NaPO containing 0.9% NaCl (PBS, 7.4) followed by 30 ml of fixative containing 4% paraformaldehyde from a pH regulator of 0.1 M NaPO 4 (PB, 7.4). The brains were removed, cryoprotected for 48 hours in PBS containing 20% sucrose, frozen in a bed of dry powdered ice, and then cut into sections of 35 micras in a sliding microtome. Sequential sections of 1 in 18 series were collected in 0.1 M PB and processed for immunohistochemistry as described below. For immunohistochemical analysis, sections were incubated for one hour in 50 mM Tris buffer, pH 7.4 containing 0.9% NaCl (TBS) and 0.1% Triton X-100. then the sections were washed in TBS and incubated for one hour in an antibody vehicle (4% goat or horse serum, 0.1% Triton X-100 in TBS).

After additional washes in TBS, the sections were incubated overnight at 4 ° C in vehicle containing the primary antibody. After incubation overnight in the primary antibody, the sections were washed in TBS, incubated for one hour in vehicle containing anti-mouse or goat anti-rabbit horse antibody with biotin (according to the manufacturer, Vector Labs, Buriingame, CA), washed in TBS and then incubated for one hour in TBS containing ABC reagent / horseradish peroxidase (Vector Labs, Buriingame, CA.). After additional washes in TBS the immuno-localization products were visualized by developing the sections for 3-5 minutes in 50 mM Tris buffer (pH 7.6) containing 0.04% diaminobenzidine (DAB) and 0.003% H2O2. The sections were then washed in the Tris buffer, mounted on slides and dehydrated and covered with coverslips using DPX (Fluka, Ronkokoma, N.Y.) Antibodies AT-8: Used at 1: 1, 000 (200 ng / ml) mouse monoclonal which recognizes to be 202 and thr 20d of human phosphorylated tau (Innogenetics, Inc. Zwij + indrech (Belgium). PHF-13: Used at 1: 20,000, monoclonal mouse that recognizes being 396 and thr 404 of human phosphorylated tau (gift from V. Lee, University of Pennsylvania) Anti-TAU: Used at 1: 7,000, rabbit polyclonal that recognizes total tau ( Accurate Chemical and Scientific Corp Westbury, NY) SMI 34: Used at 1: 1,000, monoclonal mouse that recognizes phosphorylated neurofilament H (Sternberger Monoclonals, Inc., Lutherville MD) Brains from transgenic 4-month-old mice and wild type were compared in terms of the presence of tau and neurofilament phosphoepitopes by immunohistochemistry.

PHF-13, monoclonal antibodies that recognize Ser-202 and Thr-20d and Ser-396, 404 phosphorylated, respectively, marked the neurons in the amygdala (Figures 2 and 4, respectively), thalamus / hypothalamus and cortex adjacent to the outer capsule (not shown) of the transgenic animals but not in the corresponding regions of the wild-type animals (Figures 3 and d).

In addition, staining for total tau revealed numerous cell bodies in the p2d transgenic animals (Figure 6) although most of the axons and a few cell bodies were labeled in the wild-type mice (Figure 7). A monoclonal antibody (SMI34) that recognizes neurofilament Phosphorylated H also showed increased immunostaining in these three brain regions and in the spinal cord of the transgenic mice (the amygdala is shown in Figure 8), but not in the wild type (Figure 9). It is known that all these three markers are increased in the Alzheimer's brain in relation to the control with the same age. In the Alzheimer's brain, pathological changes in neurons containing altered tau proteins have been classically identified using silver-based staining methods. It was found that, in the transgenic mice of this invention, neurons in the same three brain regions and in the spinal cord show positive labeling using the modified Bielschhowsky silver stain (amygdala, Figure 10). This staining was again absent in the wild-type control animals (Figure 11). Increases in silver staining and phosphorylated neurofilament H were also detected in the obviously elongated axons in the spinal cord of the transgenic mice (figures 12 and 13 respectively), whereas in the wild-type mice, the axons were of the expected diameter and the neurofilament staining was normal (figure 14). Regarding the immunoreactivity of AT-8, most of the marked neurons in the amygdala presented well defined soma, with dense staining, frequently with a dystrophic, swollen prominence and a contorted axon (figure 2). Less commonly, a diffuse labeling of unidentified cells was observed, and cells that have astroglial or microglial morphology were occasionally marked. In the cortex adjacent to the outer capsule, most staining with AT-8 was found in intensely marked neuronal cell bodies, often accompanied by axons when present in the plane of the section. 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Claims (4)

NOVELTY OF THE INVENTION CLAIMS

1. - A recombinant DNA comprising a rat neuron-specific enolase promoter operably linked to a p2d coding sequence of the coding sequence of the human cdkd gene.

2. The recombinant DNA according to claim 1, further characterized in that said sequence coding for said fragment of p2d has the characteristics of genomic DNA.

3. The recombinant DNA according to claim 1, further characterized in that said sequence coding for said fragment of p2d has the characteristics of cDNA.

4. The recombinant DNA according to claim 1, further characterized in that said sequence is of SEQ ID NO: 4 d.- A vector comprising the recombinant DNA according to claim 1. 6. A vector comprising the Recombinant DNA according to claim 2. 7. A vector comprising the recombinant DNA according to claim 3. 8. A line of eukaryotic cells comprising the recombinant DNA according to claim 1. 9.- A eukaryotic cell line comprising the recombinant DNA according to claim 2. 10. A line of eukaryotic cells comprising the recombinant DNA d according to claim 3. 11. A transgenic non-human mammal and progeny thereof, whose germ cells and somatic cells express the recombinant DNA according to claim 1. 12.- A transgenic non-human mammal or progeny thereof, Or whose germ cells and somatic cells express the recombinant DNA according to claim 2. 13. A transgenic non-human mammal or progeny thereof, whose germ cells and somatic cells express the recombinant DNA according to claim 3. d 14 .- The transgenic non-human animal, or a progeny thereof, according to claim 12, which is a mouse. 1d.- The transgenic non-human animal, or a progeny thereof, according to claim 13, which is a mouse. 16. The transgenic non-human animal, or a progeny of the same, according to claim 14, which is a mouse. 17. The use of an inhibitor of a p25 fragment for the manufacture of a medicament for treating an animal suffering from a disease characterized by the expression of a p2d fragment of a human cdkd gene. 18. A method for determining the ability of a compound to inhibit the expression of a p2d fragment of a human cdkd gene, characterized in that it comprises the steps of: a. creating a transgenic non-human animal by stably incorporating into the embryonic stem cells of said animal, the recombinant DNA of claim 1; b. developing said embryonic stem cells in a mature transgenic non-human animal; c. administering to said transgenic non-human animal the compound of interest; and d. measuring the inhibition of said fragment of p2d by said compound. 19. A method for generating data to determine the ability of a compound to inhibit the expression of a p2d fragment of a human cdkd gene, characterized in that it comprises the steps of: a. creating a transgenic non-human animal by stably incorporating into the embryonic stem cells of said animal, the recombinant DNA of claim 1; b. developing said embryonic stem cells in a mature transgenic non-human animal; c. administering to said transgenic non-human animal the compound of interest; d. measuring the inhibition of said fragment of p25 by said compound; and e. using the data derived from said inhibition to synthesize compounds capable of inhibiting said p2d fragment.