WO2014053692A1 - Animal model with cognitive deficit, method for obtaining same, and applications - Google Patents

Animal model with cognitive deficit, method for obtaining same, and applications Download PDF

Info

Publication number
WO2014053692A1
WO2014053692A1 PCT/ES2013/070693 ES2013070693W WO2014053692A1 WO 2014053692 A1 WO2014053692 A1 WO 2014053692A1 ES 2013070693 W ES2013070693 W ES 2013070693W WO 2014053692 A1 WO2014053692 A1 WO 2014053692A1
Authority
WO
WIPO (PCT)
Prior art keywords
megalin
mice
animal model
animal
model
Prior art date
Application number
PCT/ES2013/070693
Other languages
Spanish (es)
French (fr)
Inventor
Eva María CARRO DÍAZ
Desiree Antequera Tienda
Consuelo PASCUAL PÉREZ
Original Assignee
Fundación Para La Investigación Biomédica Del Hospital 12 De Octubre
Centro De Investigación Biomédica En Red De Enfermedades Neurodegenerativas (Ciberned)
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fundación Para La Investigación Biomédica Del Hospital 12 De Octubre, Centro De Investigación Biomédica En Red De Enfermedades Neurodegenerativas (Ciberned) filed Critical Fundación Para La Investigación Biomédica Del Hospital 12 De Octubre
Publication of WO2014053692A1 publication Critical patent/WO2014053692A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knockout animals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/20—Animal model comprising regulated expression system
    • A01K2217/206—Animal model comprising tissue-specific expression system, e.g. tissue specific expression of transgene, of Cre recombinase
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • A01K2267/0312—Animal model for Alzheimer's disease

Definitions

  • the present invention falls within the field of biotechnology. Specifically it refers to the development of nonhuman animals useful as models of human diseases related to cognitive deficits, and more specifically as a model of Alzheimer's.
  • Alzheimer's disease is one of the most important diseases in biomedical research. It is one of the neurodegenerative diseases that has the most impact on society. This disease causes a cognitive deterioration of the patient, and can end up developing in such a way that it can lead to the death of the patient. Due to the progressive aging of the population in industrialized countries, the mortality rate due to this disease is rising. The existence of a low number of useful drugs has led to a great development of the number of investigations carried out in order to know the pathogenesis of the disease. In addition, the number of compounds tested has been increased with the intention of finding treatments for the disease. However, at present it is still necessary to investigate in greater depth the pathophysiological mechanisms that the disease entails, and the search for drugs that allow a remission of the disease and an improvement in the quality of life of patients.
  • megalin / LRP2 a member of the family of low-density lipoprotein (LDL) receptor genes, is involved in the cellular uptake of various macromolecules, including numerous neurotrophic factors vital to the normal functioning of the brain.
  • Megalin also known as LRP-2 and glycoprotein 330, is the largest member of the low density lipoprotein receptor (LDLR) family, which also includes VLDLR, ApoER2 / LRP8, LRP1, LRP1 B, SorLA / LR1 1, LRP5, LRP6 and MEGF7 (Jaeger and Pietrzik, 2008, Curr Alzheimer Res 5 (1): 15-25; May et al., 2007, Ann Med 39 (3): 219-28).
  • LDLR low density lipoprotein receptor
  • Megalin is expressed in several absorbent epithelia, including renal proximal tubules, visceral vitelline vesicle, intestinal brush border, thyroid follicular cells, epididymis, male and female reproductive tracts and inner ear epithelium (Christensen and Birn, 2002, Nat Rev Mol Cell B ⁇ ol 3 (4): 256-66; Argraves and Morales, 2004, Mol Reprod Dev 69 (4): 419-27; Moestrupt and Verroust, 2001, Annu Rev Nutr 21: 407-28; Van Praet, 2003, Mol Reprod Dev 64 (2): 129-35).
  • megalin has been described in astrocytes (Bento-Abreu et al, 2008, J Neurochem 106 (3): 1 149-59), culture of granular neurons in the cerebellum (Ambj0rn et al, 2008, J Neurochem 104 (1 ): 21-37), sensory neurons (Fleming et al, 2009, J Neurosci. 29 (10): 3220-32) and cortical neurons (Chung et al, 2008, J Biol Chem. 283 (22): 15349-58 ; Alvira-Botero et al., 2010, Mol Cell Neurosci. 45 (3): 306-15).
  • megalin deficient animals revealed serious abnormalities in the development of the kidneys, lungs and CNS, consistent with the pattern of protein expression.
  • This phenotype is consistent with the role of megalin as an endocytic receptor that mediates the cellular uptake of essential nutrients, possibly derived from cholesterol lipoproteins, from the amniotic fluid in the rapid division of the neuroepithelium before the establishment of a complete circulatory system in the embryo ( Willnow et al, 1996, Proc Nati Acad Sci US A. 93 (16): 8460-4).
  • the present invention faces the need to find animal models of neurodegenerative diseases that allow the etiopathogenic study of these diseases, as well as the need for models that allow the analysis of drugs for the prevention and / or treatment of human disease.
  • the inventors demonstrate that by eliminating the expression of megalin (LRP-2) specifically in the endothelium of the cerebral capillaries of non-human animals, an animal model that presents cognitive deficit is obtained, and that reproduces behaviors similar to human diseases that present with cognitive deficit such as Alzheimer's.
  • LRP-2 megalin
  • a specific endothelial cell promoter and since the megalin in the endothelium is expressed exclusively in the cerebral capillaries, it is possible to eliminate the presence of megalin specifically in these cerebral capillaries, while It is still present in the other tissues that express it regularly.
  • the model thus generated presents behavioral alterations similar to those of other previously described models such as those of amyloidosis models.
  • the elimination of megalin must be carried out exclusively in the endothelium of the cerebral capillaries since otherwise, since megalin is a receptor present in various tissues, other defects would occur in the animal generated so that it would not present the same utility as in the present invention.
  • the absence of megalin in all tissues can cause that the correct development of these tissues or of the animal does not occur, which can cause premature death thereof due to developmental defects and therefore it is not possible for example the use of adult animals or studies that need long waiting times, such as the analysis of drug effects.
  • the deficiencies in the development of these tissues cause animals with absence of megalin in tissues other than vascular capillaries to present different utilities to the model of the present invention.
  • the model of the invention the animal can reach adulthood, and therefore it is useful for behavioral studies or for the identification and / or evaluation of compounds or compositions for the prevention or treatment of diseases related to a cognitive deficit .
  • a first aspect of the invention relates to a non-human animal model, hereinafter animal model of the invention, characterized by the absence of megalin located specifically in the endothelium of the cerebral capillaries.
  • the model of the invention is more useful when the model is a non-human mammal since it has characteristics more similar to those of human disease than in other types of animals. Therefore in one preferred embodiment of the first aspect of the invention the non-human animal is a non-human mammal.
  • the non-human mammal is a rodent or a primate. In an even more preferred embodiment of this aspect of the invention, the non-human mammal is a mouse.
  • the model of the invention can have any genetic background, although it is advantageous for said animal to be a normal animal that has no diagnosed pathologies.
  • abnormal applied to animals, as used in the present invention, refers to animals that lack transgenes or genetic alterations that could be involved in the pathogenesis of neurodegenerative diseases.
  • removal can be carried out by a transgenesis process in a way that does not allow the expression of the LRP-2 (low density lipoprotein receptor-related protein 2) or megalin gene in the cerebral capillaries.
  • This process can be carried out, for example, but not limited, as illustrated in the examples of the present invention, by the Cre / lox system under a specific tissue promoter, in this case endothelial cells such as the Tie-promoter. 2.
  • the Cre / lox system under a specific tissue promoter, in this case endothelial cells such as the Tie-promoter. 2.
  • the animals thus generated will present absence of megalin in the cerebral capillaries, maintaining the expression of this protein in the rest of tissues in which it is expressed in a normal animal such as, for example, choroidal plexus epithelial cells, neurons , astrocytes, epithelial cells of proximal tubules of the kidney, vitelline visceral sac, intestinal brush border, thyroid follicular cells, epididymis, reproductive tract and / or epithelium of the inner ear. Therefore, in another preferred embodiment of this aspect of the invention, the non-human animal is a transgenic animal.
  • a preferred embodiment of the first aspect of the invention relates to a nonhuman animal model of cognitive deficit characterized by the absence of megalin located specifically in the endothelium of the cerebral capillaries useful as an experimental model of a neurodegenerative disease.
  • a more preferred embodiment refers to a nonhuman animal model of cognitive deficit characterized by the absence of megalin located specifically in the endothelium of the cerebral capillaries useful as an experimental model of a neurodegenerative disease, where the neurodegenerative disease is Alzheimer's.
  • Another aspect of the invention relates to a process for obtaining the animal model of the invention, hereafter referred to as the method of invention, which comprises the elimination of megalin specifically in the endothelium of the cerebral capillaries.
  • the elimination of megalin can occur for example, but without being limited by transgenesis so that there is an inhibition of the expression of megalin in the endothelium of the cerebral capillaries specifically, without affecting cells of other tissues where it also occurs gene expression In this way the animal model of the invention can be obtained. Therefore, in a preferred embodiment of the method of the invention, the elimination of megalin is produced by transgenesis.
  • transgenesis refers to any technique or procedure that allows the integration into a series of cells of a living organism of an exogenous gene, or "transgene”, without affecting all of the cells of said organism, and which confers said cells and the organism that carries them a new biological property.
  • Said transgene or exogenous gene refers to a DNA that is not normally resident, nor present in the cell to be transformed.
  • the model of the invention can be obtained by different transgenesis strategies known to the expert. However, it is desirable that the process can be a controlled process to allow a better achievement of the model as well as a better use of the animal. Thus, as demonstrated in the examples of the invention, one of the possible strategies to be used is the use of the "Cre / lox" system controlled by a specific tissue promoter.
  • the model of the invention can be obtained by crossing animals that have substituted the endogenous Megalin sequence by the Megalin sequence flanked by the lox sequences, with animals having the Cre bacterial recombinase directed by a specific endothelial tissue promoter such as the Tie-2 promoter.
  • Cre recombinase is expressed in the endothelial tissue, and therefore only the capillaries Cerebral that is where megalin is expressed, will present absence of protein expression while in the rest of tissues this expression will not be affected. Therefore, in a more preferred embodiment of the method of the invention, the elimination of megalin is produced by transgenesis by the Cre / lox system. In an even more preferred embodiment, the Cre / lox system is controlled by the Tie2 promoter.
  • the model of the invention reproduces the behaviors that occur in diseases in which there is a cognitive deficit associated with dementia. Due to this, this model is useful for the study of diseases that present with cognitive deficit, such as, but not limited to, different neurodegenerative diseases such as Alzheimer's, vascular dementia, mild cognitive impairment or Parkinson's. Therefore, another aspect of the invention relates to the use of the model of the invention for the study of a neurodegenerative disease.
  • the neurodegenerative disease is a human neurodegenerative disease.
  • the neurodegenerative disease is Alzheimer's disease.
  • the model is also useful for the identification of compounds useful for the treatment of these diseases.
  • This animal model when developing common symptoms with these diseases, would be useful to test new drugs, the evolution of pharmacological treatments, their side effects, and all those aspects related to preclinical trials. Therefore, another aspect of the invention relates to the use of the model of the invention for the identification and evaluation of therapeutic compounds against a neurodegenerative disease.
  • the neurodegenerative disease is a human neurodegenerative disease.
  • the neurodegenerative disease is Alzheimer's disease.
  • Fig. 1 Shows the expression of megalin in EMD mice (Endothelial Megalin Deficient, or megalin deficient in brain capillaries), Wild-Type mice and in different tissues.
  • A) Immunohistochemistry shows megalin expression in endothelial cells in the brains of Wild-Type (WT) mice, and absence in EMD mice. The arrows indicate the expression of megalin.
  • C) Megalin expression is preserved in the choroidal plexus epithelial cells, both in wild (control) mice, and in EMD mice.
  • EMD megalin deficient mice in the cerebral capillaries; Wt: normal mice;
  • APP APP695 transgenic mice model of amyloidosis;
  • APP / Ps1 APP695 and Ps1 transgenic double mice, amyloidosis model.
  • the Cre / lox recombination system (Sauer & Henderson, 1988, Proc. Nati. Acad. Sci. USA 85,5166-5170; Sauer, 1993, Methods Enzymol. 225, has been used to carry out the model of the invention. 890-900) to eliminate the megalin gene exclusively in the cerebral capillaries.
  • the technique in this case consisted of the crossing of animals with transgenic Lox-Megalin sequences that replace the endogenous Megalin sequence, with animals that have the Cre bacterial recombinase directed by a tissue-specific promoter, in this case Tie2, specific of capillary endothelium.
  • Tie2-Cre transgenic mice mice presenting the cre gene encoding the Cre recombinase under the control of the Tie-2 promoter
  • megalin / gp330 megalin lox / lox
  • Tie2-Cre / megalin lox / lox mice were generated, which were called EMD ⁇ Endothelial Megalin Deficient, or megalin deficient in the cerebral capillaries) mice that present no expression of megalin expression in the endothelium of the cerebral capillaries.
  • Lox + / + mice which do not express Cre recombinase) bait brothers were used as controls.
  • BMECs cerebral microvasculature
  • the resulting homogenate was digested with collagenase (1 mg / mL) and DNAse (30 U / mL) in DMEM medium (containing 100 units / mL penicillin, 100 g / mL streptomycin, 50 g / mL gentamicin and 2 mM GlutaMAX-1) at 37 ° C for 40 minutes.
  • microvessel-rich precipitate was resuspended in DMEM and the mixture was seeded in culture plates previously coated with a coating buffer composed of fibronectin (0.05 mg / mL), collagen I (0.05 mg / mL) and collagen IV (0.1 mg / mL).
  • the seeded cells are incubated at 37 ° C for 24 hours in a humid environment (5% C02 / 95% air) in a 1: 1 DMEM mixture and an F12 nutrient mixture (DMEM / F-12) that has been supplemented with 20% of bovine plasma derivatives, penicillin (100 U / mL), streptomycin (100 g / mL), gentamicin (50 Mg / mL) and basic fibroblast growth factor (bFGF, 1 ng / mL).
  • DMEM / F-12 F12 nutrient mixture
  • bovine plasma derivatives penicillin (100 U / mL), streptomycin (100 g / mL), gentamicin (50 Mg / mL) and basic fibroblast growth factor (bFGF, 1 ng / mL).
  • bFGF basic fibroblast growth factor
  • BMVEC mouse cerebral capillary endothelial cells
  • Behavioral tests were performed at 6 months of age. After adaptation to human manipulation, behavioral tests were performed for 11 days.
  • the evaluation was completed by the object recognition test (days 10 and 1 1).
  • the open field was made in a maze with an area of 50 cm x 50 cm, walls 38 cm high and a central area with an area of 25 cm x 25 cm.
  • the mice were placed in a corner of the open field and evaluated in 5 min sessions for 3 days.
  • the entrances and the time elapsed in the central zone were determined as measures of behavior related to anxiety.
  • the recognition index defined as the ratio between the time spent exploring the new object and the scanning time of both objects, is used to measure memory.
  • mice A total of 62 male mice (27 EMD and 35 controls) were used in the study.
  • the behavior of two lines of transgenic mice models of amyloidosis was studied, transgenic mice that overexpress the human APP695 peptide, and double transgenic mice APP / Ps1, a cross between mice that overexpress the human APP695 peptide and the Mutant form Ps1 (M146L).
  • the behavior of the EMD mice and controls of 6 months of age was evaluated.
  • T-labyrinth test a significant increase in latency was found with respect to control mice that was similar to that shown by amyloidosis models, APP / PS1 mice and APP mice (Figure 2A).

Abstract

The invention relates to an animal model with cognitive deficit, exhibiting absence of megalin specifically in the endothelium of the brain capillaries. The invention also relates to the method for obtaining same and to the use thereof as a model of neurodegenerative diseases, more concretely, in Alzheimer disease.

Description

MODELO ANIMAL DE DEFICIT COGNITIVO. PROCEDIMIENTO DE  ANIMAL MODEL OF COGNITIVE DEFICIT. PROCEDURE OF
OBTENCIÓN Y APLICACIONES  OBTAINING AND APPLICATIONS
DESCRIPCIÓN DESCRIPTION
La presente invención se encuadra en el campo de la biotecnología. Específicamente se refiere al desarrollo de animales no humanos útiles como modelos de enfermedades humanas relacionadas con déficit cognitivo, y más específicamente como modelo de Alzheimer. The present invention falls within the field of biotechnology. Specifically it refers to the development of nonhuman animals useful as models of human diseases related to cognitive deficits, and more specifically as a model of Alzheimer's.
ESTADO DE LA TÉCNICA STATE OF THE TECHNIQUE
La enfermedad de Alzheimer es una de las enfermedades que mayor importancia presenta en la investigación biomédica. Se trata de una de las enfermedades neurodegenerativas que más impacto tiene en la sociedad. Esta enfermedad provoca un deterioro cognitivo del paciente, y puede acabar desarrollándose de tal forma que puede llevar a la muerte del paciente. Debido al progresivo envejecimiento de la población en los países industrializados, la tasa de mortalidad debida a esta enfermedad se encuentra en ascenso. La existencia de un bajo número de fármacos útiles ha provocado un gran desarrollo del número de investigaciones realizadas en aras de conocer la etiopatogénesis de la enfermedad. Además, se ha aumentado el número de compuestos ensayados con la intención de encontrar tratamientos para la enfermedad. Sin embargo, en la actualidad sigue siendo necesaria la investigación en mayor profundidad de los mecanismos etiopatológicos que conlleva la enfermedad, y la búsqueda de fármacos que permitan una remisión de la enfermedad y una mejora en la calidad de vida de los pacientes. Alzheimer's disease is one of the most important diseases in biomedical research. It is one of the neurodegenerative diseases that has the most impact on society. This disease causes a cognitive deterioration of the patient, and can end up developing in such a way that it can lead to the death of the patient. Due to the progressive aging of the population in industrialized countries, the mortality rate due to this disease is rising. The existence of a low number of useful drugs has led to a great development of the number of investigations carried out in order to know the pathogenesis of the disease. In addition, the number of compounds tested has been increased with the intention of finding treatments for the disease. However, at present it is still necessary to investigate in greater depth the pathophysiological mechanisms that the disease entails, and the search for drugs that allow a remission of the disease and an improvement in the quality of life of patients.
Para el estudio de la enfermedad y de fármacos útiles para su tratamiento o para la atenuación de síntomas, se hace necesaria la búsqueda de modelos animales que reproduzcan las características de la enfermedad presentes en la enfermedad en seres humanos. Para ello se han desarrollado diferentes modelos animales como por ejemplo diversos animales transgénicos portadores de las distintas mutaciones encontradas en pacientes de Alzheimer familiar, tales como presenilinas y amiloide (Hock & Lamb, 2001 , Trends Genet. 17(10):S7-12.). Un inconveniente muy importante es que si bien estos animales mutantes presentan varios de los síntomas de la enfermedad de Alzheimer, ninguno de ellos exhibe todo el espectro de cambios patológicos asociados a la misma (Richardson et al., 2002, ILAR J. 43(2):89-99). En un intento de solucionar este problema se han cruzado entre sí cepas de ratones transgénicos con las distintas mutaciones que recrean cada una de ellas distintos aspectos de la enfermedad para así lograr un modelo que se asemeje mejor a la patología humana (Phinney et al., 2003, Neurol Res. 25(6):590-600). For the study of the disease and of drugs useful for its treatment or for the attenuation of symptoms, it is necessary to search for animal models that reproduce the characteristics of the disease present in the disease in humans. For this they have developed different animal models such as various transgenic animals carrying the different mutations found in familial Alzheimer's patients, such as presenilins and amyloid (Hock & Lamb, 2001, Trends Genet. 17 (10): S7-12.). A very important drawback is that although these mutant animals have several of the symptoms of Alzheimer's disease, none of them exhibit the full spectrum of pathological changes associated with it (Richardson et al., 2002, ILAR J. 43 (2 ): 89-99). In an attempt to solve this problem, strains of transgenic mice have been crossed with the different mutations that recreate each of them different aspects of the disease in order to achieve a model that best resembles human pathology (Phinney et al., 2003, Neurol Res. 25 (6): 590-600).
A pesar de estos esfuerzos, aun resulta necesaria la elaboración de nuevos modelos que permitan estudiar las enfermedades neurológicas desde diferentes enfoques y analizando la implicación de diferentes elementos. Despite these efforts, it is still necessary to develop new models that allow the study of neurological diseases from different approaches and analyzing the implication of different elements.
Por otro lado se ha comprobado que la megalina/LRP2, miembro de la familia de genes receptores de lipoproteínas de baja densidad (LDL), está implicada en la captación celular de diversas macromoléculas, incluyendo numerosos factores neurotróficos vitales para el normal funcionamiento del cerebro. La megalina, también conocida como LRP-2 y glicoproteína 330, es el mayor miembro de la familia de receptores de lipoproteínas de baja densidad (LDLR), que también incluye VLDLR, ApoER2/LRP8, LRP1 , LRP1 B, SorLA/LR1 1 , LRP5, LRP6 y MEGF7 (Jaeger y Pietrzik, 2008, Curr Alzheimer Res 5(1 ):15-25; May et al., 2007, Ann Med 39(3):219-28). La megalina se expresa en varios epitelios absorbentes, incluyendo túbulos proximales renales, vesícula vitelina visceral, borde de cepillo intestinal, células foliculares de tiroides, epidídimo, tractos reproductivos masculinos y femeninos y epitelio del oído interno (Christensen y Birn, 2002, Nat Rev Mol Cell Bíol 3(4):256-66; Argraves y Morales, 2004, Mol Reprod Dev 69(4):419-27; Moestrupt y Verroust, 2001 , Annu Rev Nutr 21 :407-28; Van Praet, 2003, Mol Reprod Dev 64(2): 129-35). En el sistema nervioso central (CNS) la megalina fue descrita por primera vez en los capilares del cerebro, revestimiento ependimario de las paredes ventriculares y plexos coroideos (Zlokovic et al., 1996, Proc Nati Acad Sci U S A. 93(9):4229-34 ; Zheng y Zhao, 2002, Methods Mol Biol 188:99-1 14; Chun et al., 1999, Exp Neurol 157(1 ):194-201 ; Kounnas et al, 1994, In Vivo 8(3):343-51 ) y más tarde en progenitores neuronales en la médula espinal de embriones de ratón (Wicher et al, 2005, J Comp Neurol 492(2): 123-31 ) y oligodendrocitos de médula espinal de ratón postnatal (Wicher et al, 2006, J Neuroscí Res 83(5):864-73). Más recientemente, la megalina ha sido descrita en astrocitos (Bento-Abreu et al, 2008, J Neurochem 106(3): 1 149-59), cultivo de neuronas granulares del cerebelo (Ambj0rn et al, 2008, J Neurochem 104(1 ):21 -37), neuronas sensoriales (Fleming et al, 2009, J Neurosci. 29(10):3220-32) y neuronas corticales (Chung et al, 2008, J Biol Chem. 283(22): 15349-58; Alvira- Botero et al., 2010, Mol Cell Neurosci. 45(3):306-15). On the other hand, it has been proven that megalin / LRP2, a member of the family of low-density lipoprotein (LDL) receptor genes, is involved in the cellular uptake of various macromolecules, including numerous neurotrophic factors vital to the normal functioning of the brain. Megalin, also known as LRP-2 and glycoprotein 330, is the largest member of the low density lipoprotein receptor (LDLR) family, which also includes VLDLR, ApoER2 / LRP8, LRP1, LRP1 B, SorLA / LR1 1, LRP5, LRP6 and MEGF7 (Jaeger and Pietrzik, 2008, Curr Alzheimer Res 5 (1): 15-25; May et al., 2007, Ann Med 39 (3): 219-28). Megalin is expressed in several absorbent epithelia, including renal proximal tubules, visceral vitelline vesicle, intestinal brush border, thyroid follicular cells, epididymis, male and female reproductive tracts and inner ear epithelium (Christensen and Birn, 2002, Nat Rev Mol Cell Bíol 3 (4): 256-66; Argraves and Morales, 2004, Mol Reprod Dev 69 (4): 419-27; Moestrupt and Verroust, 2001, Annu Rev Nutr 21: 407-28; Van Praet, 2003, Mol Reprod Dev 64 (2): 129-35). In the central nervous system (CNS) megalin was described for the first time in the capillaries of the brain, ependymal lining of the ventricular walls and choroid plexuses (Zlokovic et al., 1996, Proc Nati Acad Sci US A. 93 (9): 4229-34; Zheng and Zhao, 2002, Methods Mol Biol 188: 99-1 14; Chun et al., 1999, Exp Neurol 157 (1): 194-201; Kounnas et al, 1994, In Vivo 8 (3) : 343-51) and later in neuronal progenitors in the spinal cord of mouse embryos (Wicher et al, 2005, J Comp Neurol 492 (2): 123-31) and postnatal mouse spinal cord oligodendrocytes (Wicher et al , 2006, J Neuroscí Res 83 (5): 864-73). More recently, megalin has been described in astrocytes (Bento-Abreu et al, 2008, J Neurochem 106 (3): 1 149-59), culture of granular neurons in the cerebellum (Ambj0rn et al, 2008, J Neurochem 104 (1 ): 21-37), sensory neurons (Fleming et al, 2009, J Neurosci. 29 (10): 3220-32) and cortical neurons (Chung et al, 2008, J Biol Chem. 283 (22): 15349-58 ; Alvira-Botero et al., 2010, Mol Cell Neurosci. 45 (3): 306-15).
El análisis de animales deficientes de megalina reveló graves anormalidades en el desarrollo de los ríñones, pulmones y CNS, consistente con el patrón de expresión de la proteína. Este fenotipo es consistente con el papel de la megalina como receptor endocítico que media la captación celular de nutrientes esenciales, posiblemente derivado de lipoproteínas de colesterol, del líquido amniótico en la rápida división del neuroepitelio antes del establecimiento de un sistema circulatorio completo en el embrión (Willnow et al, 1996, Proc Nati Acad Sci U S A. 93(16):8460-4). The analysis of megalin deficient animals revealed serious abnormalities in the development of the kidneys, lungs and CNS, consistent with the pattern of protein expression. This phenotype is consistent with the role of megalin as an endocytic receptor that mediates the cellular uptake of essential nutrients, possibly derived from cholesterol lipoproteins, from the amniotic fluid in the rapid division of the neuroepithelium before the establishment of a complete circulatory system in the embryo ( Willnow et al, 1996, Proc Nati Acad Sci US A. 93 (16): 8460-4).
Por ello, la implicación de esta proteína en enfermedades no ha podido ser estudiado ya que los animales deficientes en ella no presentan un correcto desarrollo y presentan graves anormalidades en el desarrollo de diversos tejidos. Therefore, the implication of this protein in diseases has not been able to be studied since the animals deficient in it do not present a correct development and present serious abnormalities in the development of various tissues.
DESCRIPCIÓN DE LA INVENCIÓN La presente invención se enfrenta a la necesidad de encontrar modelos animales de enfermedades neurodegenerativas que permitan el estudio etiopatogénico de estas enfermedades, así como a la necesidad de modelos que permitan el análisis de fármacos para la prevención y/o el tratamiento de la enfermedad humana. DESCRIPTION OF THE INVENTION The present invention faces the need to find animal models of neurodegenerative diseases that allow the etiopathogenic study of these diseases, as well as the need for models that allow the analysis of drugs for the prevention and / or treatment of human disease.
En la presente invención los inventores demuestran como mediante la eliminación de la expresión de megalina (LRP-2) específicamente en el endotelio de los capilares cerebrales de animales no humanos, se obtiene un modelo animal que presenta déficit cognitivo, y que reproduce comportamientos similares a enfermedades humanas que cursan con déficit cognitivo como por ejemplo el Alzheimer. Mediante el sistema Cre/lox bajo el promotor Tie2, un promotor específico de células endoteliales, y dado que la megalina en el endotelio se expresa exclusivamente en los capilares cerebrales, se consigue eliminar la presencia de megalina de forma específica en estos capilares cerebrales, mientras sigue estando presente en los demás tejidos que la expresan de forma habitual. El modelo así generado, tal y como se muestra en los ejemplos, presenta alteraciones comportamentales similares a los de otros modelos previamente descritos como por ejemplo las de modelos de amiloidosis. La eliminación de megalina se ha de realizar de forma exclusiva en el endotelio de los capilares cerebrales ya que en caso contrario, dado que la megalina es un receptor presente en diversos tejidos, se producirían otros defectos en el animal generado de forma que no presentaría la misma utilidad que en la presente invención. Además, la ausencia de megalina en todos los tejidos puede provocar que no se produzca el correcto desarrollo de estos tejidos o del animal lo que puede provocar la muerte prematura del mismo por defectos en el desarrollo y por tanto que no sea posible por ejemplo el uso de animales adultos o de estudios que necesiten tiempos de espera largos, como por ejemplo el análisis de los efectos de fármacos. Además las deficiencias en el desarrollo de estos tejidos provocan que los animales con ausencia de megalina en tejidos diferentes a los capilares vasculares presenten utilidades diferentes al modelo de la presente invención. Como se muestra en los ejemplos, el modelo de la invención el animal puede llegar a la edad adulta, y por ello presenta utilidad para los estudios comportamentales o para la identificación y/o evaluación de compuestos o composiciones para la prevención o el tratamiento de enfermedades relacionadas con un déficit cognitivo. In the present invention, the inventors demonstrate that by eliminating the expression of megalin (LRP-2) specifically in the endothelium of the cerebral capillaries of non-human animals, an animal model that presents cognitive deficit is obtained, and that reproduces behaviors similar to human diseases that present with cognitive deficit such as Alzheimer's. Through the Cre / lox system under the Tie2 promoter, a specific endothelial cell promoter, and since the megalin in the endothelium is expressed exclusively in the cerebral capillaries, it is possible to eliminate the presence of megalin specifically in these cerebral capillaries, while It is still present in the other tissues that express it regularly. The model thus generated, as shown in the examples, presents behavioral alterations similar to those of other previously described models such as those of amyloidosis models. The elimination of megalin must be carried out exclusively in the endothelium of the cerebral capillaries since otherwise, since megalin is a receptor present in various tissues, other defects would occur in the animal generated so that it would not present the same utility as in the present invention. In addition, the absence of megalin in all tissues can cause that the correct development of these tissues or of the animal does not occur, which can cause premature death thereof due to developmental defects and therefore it is not possible for example the use of adult animals or studies that need long waiting times, such as the analysis of drug effects. In addition, the deficiencies in the development of these tissues cause animals with absence of megalin in tissues other than vascular capillaries to present different utilities to the model of the present invention. As shown in the examples, the model of the invention the animal can reach adulthood, and therefore it is useful for behavioral studies or for the identification and / or evaluation of compounds or compositions for the prevention or treatment of diseases related to a cognitive deficit .
La ausencia de megalina presenta como ventaja frente a otros métodos de inhibición de la proteína o de bloqueo funcional que no existe un posible efecto residual de la parte de la proteína que no sea bloqueada y que compense la falta de actividad de la proteína y por tanto enmascare los efectos del bloqueo o la inhibición. The absence of megalin has as an advantage over other methods of protein inhibition or functional blockage that there is no possible residual effect of the part of the protein that is not blocked and that compensates for the lack of activity of the protein and therefore mask the effects of blockage or inhibition.
Por todo ello, un primer aspecto de la invención se refiere a un modelo animal no humano, de ahora en adelante modelo animal de la invención, caracterizado por la ausencia de megalina localizada específicamente en el endotelio de los capilares cerebrales. Therefore, a first aspect of the invention relates to a non-human animal model, hereinafter animal model of the invention, characterized by the absence of megalin located specifically in the endothelium of the cerebral capillaries.
Se entiende por "ausencia de megalina localizada específicamente en el endotelio de los capilares cerebrales" la ausencia de megalina en este tejido mientras la megalina se sigue encontrando expresada en el resto de los tejidos que la presentan de forma habitual, incluyendo por ejemplo, a nivel cerebral: células epiteliales de plexos coroideos, neuronas y astrocitos y a nivel extracerebral: células epiteliales de túbulos proximales del riñon, saco visceral vitelino, borde de cepillo intestinal, las células foliculares tiroideas, epidídimo, tractos reproductivos masculinos y femeninos y epitelio del oído interno. It is understood by "absence of megalin located specifically in the endothelium of the cerebral capillaries" the absence of megalin in this tissue while megalin is still expressed in the rest of the tissues that present it in a habitual way, including for example, at the level cerebral: epithelial cells of choroid plexus, neurons and astrocytes and extracerebral level: epithelial cells of proximal tubules of the kidney, vitelline visceral sac, border of intestinal brush, thyroid follicular cells, epididymis, male and female reproductive tracts and epithelium of the inner ear.
En la presente invención los términos "megalina", "LRP-2", "glicoproteína 330" o "gp330", se refieren a la misma proteína y son intercambiables. In the present invention the terms "megalin", "LRP-2", "glycoprotein 330" or "gp330", refer to the same protein and are interchangeable.
El modelo de la invención presenta mayor utilidad cuando el modelo es un mamífero no humano ya que presenta unas características más similares a las de la enfermedad humana que en otros tipos de animales. Por tanto en una realización preferida del primer aspecto de la invención el animal no humano es un mamífero no humano. The model of the invention is more useful when the model is a non-human mammal since it has characteristics more similar to those of human disease than in other types of animals. Therefore in one preferred embodiment of the first aspect of the invention the non-human animal is a non-human mammal.
De la misma forma que en el caso anterior, dada la proximidad filogenética de los primates al ser humano, estos animales resultan un mejor modelo para la reproducción de las enfermedades producidas en humanos. Por otro lado, los modelos animales más extendidos para el estudio de enfermedades son aquellos realizados en roedores, principalmente en ratas y ratones. Esto se debe fundamentalmente a la reducción de espacio que suponen estos animales frente otros de mayor tamaño, a la facilidad de cría y manejo de los mismos y a la facilidad de obtención de un elevado número de individuos para su uso. Por todo ello, en una realización más preferida de este aspecto de la invención, el mamífero no humano es un roedor o un primate. En una realización aun más preferida de este aspecto de la invención, el mamífero no humano es un ratón. In the same way as in the previous case, given the phylogenetic proximity of primates to humans, these animals are a better model for the reproduction of diseases caused in humans. On the other hand, the most widespread animal models for the study of diseases are those made in rodents, mainly in rats and mice. This is mainly due to the reduction of space that these animals entail in front of larger ones, the ease of breeding and handling of them and the ease of obtaining a large number of individuals for use. Therefore, in a more preferred embodiment of this aspect of the invention, the non-human mammal is a rodent or a primate. In an even more preferred embodiment of this aspect of the invention, the non-human mammal is a mouse.
El modelo de la invención puede tener cualquier fondo genético, aunque es ventajoso que dicho animal sea un animal normal que no presente patologías diagnosticadas. The model of the invention can have any genetic background, although it is advantageous for said animal to be a normal animal that has no diagnosed pathologies.
El término "normal", aplicado a animal, tal como se utiliza en la presente invención, se refiere a animales que carecen de transgenes o alteraciones genéticas que podrían estar implicadas en la etiopatogenia de enfermedades neurodegenerativas. The term "normal", applied to animals, as used in the present invention, refers to animals that lack transgenes or genetic alterations that could be involved in the pathogenesis of neurodegenerative diseases.
Para dar lugar a una ausencia de megalina, la eliminación se puede llevar a cabo mediante un proceso de transgénesis de forma que no permita la expresión del gen LRP-2 (low density lipoprotein receptor-related protein 2) o megalin en los capilares cerebrales. Este proceso se puede llevar a cabo por ejemplo, aunque sin limitarse, como se ilustra en los ejemplos de la presente invención, mediante el sistema Cre/lox bajo un promotor específico de tejido, en este caso de células endoteliales como es el promotor Tie-2. Mediante este sistema, y dado que la megalina en el endotelio vascular únicamente se expresa en los capilares cerebrales, se permite la eliminación de la megalina en estos capilares sin afectar al resto de tejidos en los que se encuentra expresada. Por tanto, los animales así generados presentaran ausencia de megalina en los capilares cerebrales, manteniendo la expresión de esta proteína en el resto de tejidos en los que se expresa en un animal normal como por ejemplo, por ejemplo, células epiteliales de plexos coroideos, neuronas, astrocitos, células epiteliales de túbulos proximales del riñon, saco visceral vitelino, borde de cepillo intestinal, las células foliculares tiroideas, epidídimo, tracto reproductivos y/o epitelio del oído interno. Por todo ello, en otra realización preferida de este aspecto de la invención, el animal no humano es un animal transgénico. To result in an absence of megalin, removal can be carried out by a transgenesis process in a way that does not allow the expression of the LRP-2 (low density lipoprotein receptor-related protein 2) or megalin gene in the cerebral capillaries. This process can be carried out, for example, but not limited, as illustrated in the examples of the present invention, by the Cre / lox system under a specific tissue promoter, in this case endothelial cells such as the Tie-promoter. 2. Through this system, and since the megalin in the vascular endothelium only Expressed in the cerebral capillaries, the elimination of megalin in these capillaries is allowed without affecting the rest of the tissues in which it is expressed. Therefore, the animals thus generated will present absence of megalin in the cerebral capillaries, maintaining the expression of this protein in the rest of tissues in which it is expressed in a normal animal such as, for example, choroidal plexus epithelial cells, neurons , astrocytes, epithelial cells of proximal tubules of the kidney, vitelline visceral sac, intestinal brush border, thyroid follicular cells, epididymis, reproductive tract and / or epithelium of the inner ear. Therefore, in another preferred embodiment of this aspect of the invention, the non-human animal is a transgenic animal.
Como se demuestra en los ejemplos, los animales con ausencia de megalina específicamente en el endotelio vascular, presentan un déficit cognitivo frente a animales sin esta ausencia. Además estos animales tienen síntomas comunes y características comportamentales similares a modelos de diversas enfermedades neurodegenerativas, como por ejemplo, aunque sin limitarse, Alzheimer, demencia vascular, deterioro cognitivo leve o Parkinson. Por tanto el modelo animal no humano de la presente invención presenta utilidad como modelo experimental de enfermedad neurodegenerativa. Por ello, una realización preferida del primer aspecto de la invención se refiere a un modelo animal no humano de déficit cognitivo caracterizado por la ausencia de megalina localizada específicamente en el endotelio de los capilares cerebrales útil como modelo experimental de una enfermedad neurodegenerativa. Una realización más preferida se refiere a un modelo animal no humano de déficit cognitivo caracterizado por la ausencia de megalina localizada específicamente en el endotelio de los capilares cerebrales útil como modelo experimental de una enfermedad neurodegenerativa, donde la enfermedad neurodegenerativa es Alzheimer. As demonstrated in the examples, animals with absence of megalin specifically in the vascular endothelium, have a cognitive deficit compared to animals without this absence. In addition, these animals have common symptoms and behavioral characteristics similar to models of various neurodegenerative diseases, such as, but not limited to, Alzheimer's, vascular dementia, mild cognitive impairment or Parkinson's. Therefore the non-human animal model of the present invention has utility as an experimental model of neurodegenerative disease. Therefore, a preferred embodiment of the first aspect of the invention relates to a nonhuman animal model of cognitive deficit characterized by the absence of megalin located specifically in the endothelium of the cerebral capillaries useful as an experimental model of a neurodegenerative disease. A more preferred embodiment refers to a nonhuman animal model of cognitive deficit characterized by the absence of megalin located specifically in the endothelium of the cerebral capillaries useful as an experimental model of a neurodegenerative disease, where the neurodegenerative disease is Alzheimer's.
Otro aspecto de la invención se refiere a un procedimiento para la obtención del modelo animal de la invención, de ahora en adelante procedimiento de la invención, el cual comprende la eliminación de megalina específicamente en el endotelio de los capilares cerebrales. Another aspect of the invention relates to a process for obtaining the animal model of the invention, hereafter referred to as the method of invention, which comprises the elimination of megalin specifically in the endothelium of the cerebral capillaries.
La eliminación de megalina se puede producir por ejemplo, aunque sin limitarse mediante transgénesis de forma que se produce una inhibición de la expresión de megalina en el endotelio de los capilares cerebrales de forma específica, sin que afecte a células de otros tejidos donde también se da la expresión del gen. De esta forma se puede obtener el modelo animal de la invención. Por ello en una realización preferida del método de la invención, la eliminación de megalina se produce mediante transgénesis. The elimination of megalin can occur for example, but without being limited by transgenesis so that there is an inhibition of the expression of megalin in the endothelium of the cerebral capillaries specifically, without affecting cells of other tissues where it also occurs gene expression In this way the animal model of the invention can be obtained. Therefore, in a preferred embodiment of the method of the invention, the elimination of megalin is produced by transgenesis.
Tal como se utiliza en la presente invención el término "transgénesis" se refiere a cualquier técnica o procedimiento que permita la integración en una serie de células de un organismo vivo de un gen exógeno, o "transgén", sin afectar a la totalidad de las células de dicho organismo, y que confiere a dichas células y al organismo que las porta una nueva propiedad biológica. Dicho transgén o gen exógeno se refiere a un ADN normalmente no residente, ni presente en la célula que se pretende transformar. As used in the present invention, the term "transgenesis" refers to any technique or procedure that allows the integration into a series of cells of a living organism of an exogenous gene, or "transgene", without affecting all of the cells of said organism, and which confers said cells and the organism that carries them a new biological property. Said transgene or exogenous gene refers to a DNA that is not normally resident, nor present in the cell to be transformed.
El modelo de la invención se puede ser obtenido mediante diferentes estrategias de transgénesis conocidas por el experto. Sin embargo, resulta deseable que el proceso pueda ser un proceso controlado para permitir una mejor consecución del modelo así como un mejor uso del animal. De esta forma, tal y como se demuestra en los ejemplos de la invención, una de las posibles estrategias a utilizar es el uso del sistema "Cre/lox" controlado por un promotor específico de tejido. Por ejemplo, aunque sin limitarse, se puede obtener el modelo de la invención mediante el cruce de animales que presentan sustituida la secuencia de Megalina endógena por la secuencia de Megalina flanqueada por las secuencias lox, con animales que tengan a la recombinasa bacteriana Cre dirigida por un promotor específico del tejido endotelial como por ejemplo el promotor Tie-2. De esta forma, en el tejido endotelial se expresa la recombinasa Cre, y por tanto únicamente los capilares cerebrales que es donde se expresa la megalina, presentarán ausencia de la expresión de la proteína mientras que en el resto de tejidos está expresión no se verá afectada. Por ello, en una realización más preferida del método de la invención, la eliminación de megalina se produce mediante transgénesis por el sistema Cre/lox. En una realización aun más preferida el sistema Cre/lox se encuentra controlado por el promotor Tie2. The model of the invention can be obtained by different transgenesis strategies known to the expert. However, it is desirable that the process can be a controlled process to allow a better achievement of the model as well as a better use of the animal. Thus, as demonstrated in the examples of the invention, one of the possible strategies to be used is the use of the "Cre / lox" system controlled by a specific tissue promoter. For example, although not limited, the model of the invention can be obtained by crossing animals that have substituted the endogenous Megalin sequence by the Megalin sequence flanked by the lox sequences, with animals having the Cre bacterial recombinase directed by a specific endothelial tissue promoter such as the Tie-2 promoter. Thus, Cre recombinase is expressed in the endothelial tissue, and therefore only the capillaries Cerebral that is where megalin is expressed, will present absence of protein expression while in the rest of tissues this expression will not be affected. Therefore, in a more preferred embodiment of the method of the invention, the elimination of megalin is produced by transgenesis by the Cre / lox system. In an even more preferred embodiment, the Cre / lox system is controlled by the Tie2 promoter.
Tal y como se demuestra en los ejemplos de la presente invención, el modelo de la invención reproduce los comportamientos que se producen en enfermedades en las que existe un déficit cognitivo asociado a demencia. Debido a esto, este modelo resulta útil para el estudio de enfermedades que cursan con déficit cognitivo, como por ejemplo, aunque sin limitarse, diferentes enfermedades neurodegenerativas como el Alzheimer, demencia vascular, deterioro cognitivo leve o Parkinson. Por todo ello, otro aspecto de la invención se refiere al uso del modelo de la invención para el estudio de una enfermedad neurodegenerativa. En una realización preferida, la enfermedad neurodegenerativa es una enfermedad neurodegenerativa humana. En una realización más preferida, la enfermedad neurodegenerativa es la enfermedad de Alzheimer. As demonstrated in the examples of the present invention, the model of the invention reproduces the behaviors that occur in diseases in which there is a cognitive deficit associated with dementia. Due to this, this model is useful for the study of diseases that present with cognitive deficit, such as, but not limited to, different neurodegenerative diseases such as Alzheimer's, vascular dementia, mild cognitive impairment or Parkinson's. Therefore, another aspect of the invention relates to the use of the model of the invention for the study of a neurodegenerative disease. In a preferred embodiment, the neurodegenerative disease is a human neurodegenerative disease. In a more preferred embodiment, the neurodegenerative disease is Alzheimer's disease.
Por otro lado, debido al comportamiento del modelo animal de la invención, el modelo también resulta útil para la identificación de compuestos útiles para el tratamiento de estas enfermedades. Este modelo animal, al desarrollar síntomas comunes con dichas enfermedades, serían de utilidad para probar nuevos fármacos, la evolución de tratamientos farmacológicos, efectos secundarios de los mismos, y todas aquellos aspectos relativos a ensayos preclínicos. Por ello, otro aspecto de la invención se refiere al uso del modelo de la invención para la identificación y evaluación de compuestos terapéuticos frente a una enfermedad neurodegenerativa. En una realización preferida, la enfermedad neurodegenerativa es una enfermedad neurodegenerativa humana. En una realización más preferida, la enfermedad neurodegenerativa es la enfermedad de Alzheimer. A lo largo de la descripción y las reivindicaciones la palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, aditivos, componentes o pasos. Para los expertos en la materia, otros objetos, ventajas y características de la invención se desprenderán en parte de la descripción y en parte de la práctica de la invención. Los siguientes ejemplos y dibujos se proporcionan a modo de ilustración, y no se pretende que sean limitativos de la presente invención. On the other hand, due to the behavior of the animal model of the invention, the model is also useful for the identification of compounds useful for the treatment of these diseases. This animal model, when developing common symptoms with these diseases, would be useful to test new drugs, the evolution of pharmacological treatments, their side effects, and all those aspects related to preclinical trials. Therefore, another aspect of the invention relates to the use of the model of the invention for the identification and evaluation of therapeutic compounds against a neurodegenerative disease. In a preferred embodiment, the neurodegenerative disease is a human neurodegenerative disease. In a more preferred embodiment, the neurodegenerative disease is Alzheimer's disease. Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.
DESCRIPCIÓN DE LAS FIGURAS DESCRIPTION OF THE FIGURES
Fig. 1. Muestra la expresión de megalina en ratones EMD (Endotelial Megalin Deficient, o deficientes en megalina en los capilares cerebrales), ratones Wild- Type y en diferentes tejidos. A) Mediante inmunohistoquímica se observa expresión de megalina en las células endoteliales en los cerebros de ratones Wild-Type (WT), y ausencia en ratones EMD. Las flechas señalan la expresión de megalina. B) Se observa expresión de megalina, determinada por western blot, en lisado de células endoteliales procedentes de ratones WT y no en ratones EMD. Como control positivo se utilizó la línea de células endoteliales cerebrales b.End3. Se utilizó β-actina como control de carga. C) La expresión de megalina está preservada en las células epiteliales de los plexos coroideos, tanto en los ratones salvajes (controles), como en los ratones EMD. Fig. 1. Shows the expression of megalin in EMD mice (Endothelial Megalin Deficient, or megalin deficient in brain capillaries), Wild-Type mice and in different tissues. A) Immunohistochemistry shows megalin expression in endothelial cells in the brains of Wild-Type (WT) mice, and absence in EMD mice. The arrows indicate the expression of megalin. B) Megalin expression, determined by western blot, is observed in lysate of endothelial cells from WT mice and not in EMD mice. As a positive control, the brain endothelial cell line b.End3 was used. Β-actin was used as load control. C) Megalin expression is preserved in the choroidal plexus epithelial cells, both in wild (control) mice, and in EMD mice.
Fig. 2. Análisis de la alteración del comportamiento en los ratones. A) Test del campo abierto. B) Test del reconocimiento de objetos. C) En el test del laberinto en T. En los tres casos, EMD: ratones deficientes en megalina en los capilares cerebrales; Wt: ratones normales; APP: ratones transgénicos APP695 modelo de amiloidosis; APP/Ps1 : ratones dobles transgénicos APP695 y Ps1 , modelo de amiloidosis. Fig. 2. Analysis of behavioral disturbance in mice. A) Open field test. B) Object recognition test. C) In the T labyrinth test. In all three cases, EMD: megalin deficient mice in the cerebral capillaries; Wt: normal mice; APP: APP695 transgenic mice model of amyloidosis; APP / Ps1: APP695 and Ps1 transgenic double mice, amyloidosis model.
EJEMPLOS DE REALIZACIÓN A continuación, se ilustrará la invención mediante unos ensayos realizados por los inventores que ponen de manifiesto la efectividad del modelo de la invención como modelo animal de déficit cognitivo, así como la utilidad del método de generación del mismo. Estos ejemplos específicos que se proporcionan sirven para ilustrar la naturaleza de la presente invención y se incluyen solamente con fines ilustrativos, por lo que no han de ser interpretados como limitaciones a la invención que aquí se reivindica. Por tanto, los ejemplos descritos más adelante ilustran la invención sin limitar el campo de aplicación de la misma. EXAMPLES OF REALIZATION Next, the invention will be illustrated by tests carried out by the inventors that show the effectiveness of the model of the invention as an animal model of cognitive deficit, as well as the utility of the method of generating it. These specific examples provided serve to illustrate the nature of the present invention and are included for illustrative purposes only, and therefore should not be construed as limitations on the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting its scope of application.
EJEMPLO 1. Generación de los ratones transgénicos deficientes en megalina EXAMPLE 1. Generation of megalin-deficient transgenic mice
Para llevar a cabo el modelo de la invención se ha usado el sistema de recombinación Cre/lox (Sauer & Henderson, 1988, Proc. Nati. Acad. Sci. USA 85,5166-5170; Sauer, 1993, Methods Enzymol. 225, 890-900) para eliminar el gen de la megalina exclusivamente en los capilares cerebrales. La técnica en este caso, consistió en el cruce de animales con secuencias transgénicas Lox- Megalina que sustituyan a la secuencia Megalina endógena, con animales que tengan a la recombinasa bacteriana Cre dirigida por un promotor específico de tejido, en este caso el de Tie2, específico de endotelio capilar. Para ello se generaron a) ratones transgénicos Tie2-Cre (ratones que presentan el gen cre que codifica para la recombinasa Cre bajo el control del promotor Tie-2) y b) ratones transgénicos deficientes de megalina/gp330 (megalina lox/lox) los cuales se cruzaron. Como consecuencia de este cruce se generaron ratones Tie2-Cre/megalina lox/lox, a los que se les llamó ratones EMD {Endotelial Megalin Deficient, o deficientes en megalina en los capilares cerebrales) que presentan ausencia de la expresión de megalina en el endotelio de los capilares cerebrales. Los ratones Lox+/+ (que no expresan Cre recombinasa) hermanos de carnada fueron utilizados como controles. Al final de los experimentos, los animales fueron anestesiados con isoforano y perfundidos por vía transcardíaca con paraformaldehído en tampón fosfato (0,1 M, pH 7,4) al 4% para posteriores análisis inmunohistoquímicos. Todos los animales fueron manejados en conformidad con la Directiva 2010/63/UE, de 22 de septiembre de 2010. The Cre / lox recombination system (Sauer & Henderson, 1988, Proc. Nati. Acad. Sci. USA 85,5166-5170; Sauer, 1993, Methods Enzymol. 225, has been used to carry out the model of the invention. 890-900) to eliminate the megalin gene exclusively in the cerebral capillaries. The technique in this case consisted of the crossing of animals with transgenic Lox-Megalin sequences that replace the endogenous Megalin sequence, with animals that have the Cre bacterial recombinase directed by a tissue-specific promoter, in this case Tie2, specific of capillary endothelium. To this end, a) Tie2-Cre transgenic mice (mice presenting the cre gene encoding the Cre recombinase under the control of the Tie-2 promoter) and b) megalin / gp330 (megalin lox / lox) transgenic mice were generated which crossed. As a result of this crossing, Tie2-Cre / megalin lox / lox mice were generated, which were called EMD {Endothelial Megalin Deficient, or megalin deficient in the cerebral capillaries) mice that present no expression of megalin expression in the endothelium of the cerebral capillaries. Lox + / + mice (which do not express Cre recombinase) bait brothers were used as controls. At the end of the experiments, the animals were anesthetized with isophoran and perfused by transcardiac route with paraformaldehyde in phosphate buffer (0.1 M, pH 7.4) at 4% for subsequent immunohistochemical analyzes. All animals were managed in accordance with Directive 2010/63 / EU of September 22, 2010.
Un análisis inmunohistoquímico fue usado para comprobar que el sistema experimental bloqueaba la expresión de la proteína megalina en la microvasculatura cerebral. Para ello se incubaron secciones de cerebro de ratones EMD y controles, previamente perfundidos con paraformaldehído, con un anticuerpo primario de cabra anti-megalina (Santa Cruz Biotechnology) y se revelaron con un anticuerpo secundario fluorescente (anti-cabra AlexaFluor488, Molecular Probes). El análisis con microscopía confocal de las células endoteliales cerebrales demostró una expresión diferencial megalina en ratones EMD en comparación con ratones controles. Utilizando doble tinción de megalina con la lectina, un marcador endotelial, encontramos expresión de megalina en el endotelio de los vasos capilares cerebrales en ratones controles mientras que no se detectó señal de megalina en estas células endoteliales en ratones EMD (FiguralA). En paralelo, utilizando el mismo procesamiento histológico y con las mismas secciones cerebrales de ratones EMD y controles, se observó que la expresión de megalina se conserva en las células epiteliales de los plexos coroideos tanto en ratones controles como en ratones EMD (Figura 1 C). An immunohistochemical analysis was used to verify that the experimental system blocked the expression of the megalin protein in the cerebral microvasculature. To do this, brain sections of EMD mice and controls, previously perfused with paraformaldehyde, were incubated with a goat anti-megalin primary antibody (Santa Cruz Biotechnology) and developed with a fluorescent secondary antibody (anti-goat AlexaFluor488, Molecular Probes). Confocal microscopy analysis of brain endothelial cells demonstrated a differential megalin expression in EMD mice compared to control mice. Using double staining of megalin with lectin, an endothelial marker, we found megalin expression in the endothelium of the cerebral capillary vessels in control mice while no megalin signal was detected in these endothelial cells in EMD mice (FiguralA). In parallel, using the same histological processing and with the same brain sections of EMD and control mice, it was observed that the expression of megalin is conserved in the epithelial cells of the choroid plexus in both control and EMD mice (Figure 1 C) .
EJEMPLO 2. Cultivo primario de células endoteliales de la microvasculatura cerebral de ratón EXAMPLE 2. Primary culture of mouse cerebral microvasculature endothelial cells
Las células endoteliales de la microvasculatura cerebral (BMECs) fueron aisladas y cultivadas como se ha descrito anteriormente (Jaeger et al, 2009, J Alzheimers Dis. 17(3):553-70). Brevemente, en un experimento se aislaron cortezas cerebrales de ratones EMD de 8 semanas de edad y en un experimento paralelo se aislaron las cortezas cerebrales de los ratones controles de 8 semanas de edad. Los extractos cerebrales se limpiaron de meninges y trocearon con ayuda de un bisturí. El homogeneizado resultante fue digerido con colagenasa (1 mg/mL) y DNAsa (30 U/mL) en medio DMEM (que contiene 100 de unidades/mL de penicilina, 100 g/mL de estreptomicina, 50 g/mL de gentamicina y 2 mM GlutaMAX-l) a 37 ° C durante 40 minutos. Después de la digestión, se añadió 20% albúmina sérica bovina disuelta en DMEM, la muestra se centrifugó a 1 .000 χ g durante 20 minutos, el sobrenadante que contiene las neuronas y células gliales fue eliminado y el pellet que contiene los capilares se volvió a digerir a 37 ° C durante 30 minutos con colagenasa (1 mg/mL) y DNAsa I (30 U/mL) en DMEM). Después de la segunda digestión enzimática, el pellet separado en un gradiente de Percoll al 33% y se centrifugó a 1 .000 χ g durante 10 minutos para separar los capilares. Los capilares obtenidos fueron lavados por resuspensión en DMEM y centrifuga a 1 .000 * g durante 10 minutos. El precipitado rico en microvasos fue resuspendido en DMEM y la mezcla se sembró en placas de cultivo previamente recubiertos con un tampón de recubrimiento compuesto por fibronectina (0,05 mg/mL), colágeno I (0,05 mg/mL) y colágeno IV (0, 1 mg/mL). Las células sembradas se incuban a 37 ° C durante 24 horas en un ambiente de humedad (5% C02/95% aire) en una mezcla 1 : 1 de DMEM y una mezcla de nutrientes F12 (DMEM/F-12) que ha sido complementada con 20% de derivados de plasma bovino, penicilina (100 U/mL), estreptomicina (100 g/mL), gentamicina (50 Mg/mL) y factor de crecimiento básico de fibroblastos (bFGF, 1 ng/mL). Para eliminar otras células contaminantes, pericitos y células gliales, las BMECs fueron tratadas con puromicina (4 g/mL) durante los 2 primeros días de cultivo. El medio de cultivo fue cambiado cada día. Después de 7 días en cultivo, cuando las BMECs normalmente alcanzan el 80-90% de confluencia, las células fueron fijadas para el análisis de inmunocitoquímica o homogenizadas para determinaciones por inmunoblot. Endothelial cells of the cerebral microvasculature (BMECs) were isolated and cultured as described previously (Jaeger et al, 2009, J Alzheimers Dis. 17 (3): 553-70). Briefly, in one experiment, cerebral cortex was isolated from 8-week-old EMD mice and in a parallel experiment, cerebral cortex was isolated from mice. 8 week old controls. The brain extracts were cleaned of meninges and chopped with the help of a scalpel. The resulting homogenate was digested with collagenase (1 mg / mL) and DNAse (30 U / mL) in DMEM medium (containing 100 units / mL penicillin, 100 g / mL streptomycin, 50 g / mL gentamicin and 2 mM GlutaMAX-1) at 37 ° C for 40 minutes. After digestion, 20% bovine serum albumin dissolved in DMEM was added, the sample was centrifuged at 1000 g for 20 minutes, the supernatant containing the neurons and glial cells was removed and the pellet containing the capillaries became to be digested at 37 ° C for 30 minutes with collagenase (1 mg / mL) and DNAse I (30 U / mL) in DMEM). After the second enzymatic digestion, the pellet separated in a 33% Percoll gradient and was centrifuged at 1000 g for 10 minutes to separate the capillaries. The capillaries obtained were washed by resuspension in DMEM and centrifuged at 1 000 * g for 10 minutes. The microvessel-rich precipitate was resuspended in DMEM and the mixture was seeded in culture plates previously coated with a coating buffer composed of fibronectin (0.05 mg / mL), collagen I (0.05 mg / mL) and collagen IV (0.1 mg / mL). The seeded cells are incubated at 37 ° C for 24 hours in a humid environment (5% C02 / 95% air) in a 1: 1 DMEM mixture and an F12 nutrient mixture (DMEM / F-12) that has been supplemented with 20% of bovine plasma derivatives, penicillin (100 U / mL), streptomycin (100 g / mL), gentamicin (50 Mg / mL) and basic fibroblast growth factor (bFGF, 1 ng / mL). To eliminate other contaminating cells, pericytes and glial cells, BMECs were treated with puromycin (4 g / mL) during the first 2 days of culture. The culture medium was changed every day. After 7 days in culture, when BMECs normally reach 80-90% confluence, the cells were fixed for immunocytochemical analysis or homogenized for immunoblot determinations.
A continuación, se comprobó la eficacia del sistema experimental en cultivos primarios de células endoteliales de capilares cerebrales (BMVEC) de ratón. Como se observa en la Figura 1 B, la expresión de megalina determinada por western blot es prácticamente inexistente en el lisado celular de BMVEC procedentes de los ratones EMD en comparación con la señal observada en lisados de BMVEC de ratones controles. Como control positivo hemos utilizado una línea de células endoteliales cerebrales (b.End3) que expresó megalina. Next, the efficacy of the experimental system in primary cultures of mouse cerebral capillary endothelial cells (BMVEC) was checked. As seen in Figure 1 B, the expression of megalin determined by Western blotting is virtually non-existent in the BMVEC cell lysate from EMD mice compared to the signal observed in BMVEC lysates of control mice. As a positive control we used a line of brain endothelial cells (b.End3) that expressed megalin.
EJEMPLO 3. Desarrollo de estudios comportamentales de los ratones EMD EXAMPLE 3. Development of behavioral studies of EMD mice
Las pruebas conductuales se realizaron a los 6 meses de edad. Después de la adaptación a la manipulación humana, se realizaron pruebas de comportamientos durante 1 1 días. La alternancia espontánea, testada con el test laberinto en T, fue evaluada durante 10 días, coincidiendo desde el primer día (días 1 al 3) con el test del campo abierto. La evaluación fue completada por la prueba de reconocimiento de objetos (días 10 y 1 1 ). En el laberinto en T, se registraron el número de alteraciones y errores (entradas de los brazos visitados anteriormente) y el tiempo para completar cada sesión. El campo abierto fue hecho en un laberinto con una superficie de 50 cm x 50 cm, paredes de 38 cm de alto y una zona central con una superficie de 25 cm x 25 cm. Los ratones fueron colocados en una esquina del campo abierto y evaluados en sesiones de 5 min por 3 días. En el campo abierto se determinó las entradas y el tiempo trascurrido en la zona central como medidas de un comportamiento relacionadas con la ansiedad. El índice de reconocimiento, definido como el cociente entre el tiempo pasado explorando el objeto nuevo y el tiempo de exploración de ambos objetos, se utiliza para medir la memoria. Behavioral tests were performed at 6 months of age. After adaptation to human manipulation, behavioral tests were performed for 11 days. The spontaneous alternation, tested with the T-labyrinth test, was evaluated for 10 days, coinciding from the first day (days 1 to 3) with the open field test. The evaluation was completed by the object recognition test (days 10 and 1 1). In the labyrinth in T, the number of alterations and errors (entries of the previously visited arms) and the time to complete each session were recorded. The open field was made in a maze with an area of 50 cm x 50 cm, walls 38 cm high and a central area with an area of 25 cm x 25 cm. The mice were placed in a corner of the open field and evaluated in 5 min sessions for 3 days. In the open field, the entrances and the time elapsed in the central zone were determined as measures of behavior related to anxiety. The recognition index, defined as the ratio between the time spent exploring the new object and the scanning time of both objects, is used to measure memory.
En el estudio se utilizaron un total de 62 ratones macho (27 EMD y 35 controles). Por otra parte, se estudió el comportamiento de dos líneas de ratones transgénicos modelos de amiloidosis, los ratones transgénicos que sobre expresan el péptido humano APP695, y los ratones doble transgénicos APP/Ps1 , un cruce entre ratones que sobreexpresan el péptido humano APP695 y la forma muíante Ps1 (M146L). Se evaluó el comportamiento de los ratones EMD y controles de 6 meses de edad. En el test laberinto en T, se encontró un incremento significativo en la latencia respecto a los ratones controles que fue similar a la que exhiben los modelos de amiloidosis, los ratones APP/PS1 y los ratones APP (Figura 2A). Respecto al test del campo abierto, de nuevo los ratones EMD se comportan de forma similar a los modelos de amiloidosis. El tiempo que permanece en la zona central del laberinto es significativamente menor, tanto en el primer día como en el 2o y 3o, en comparación con los ratones controles. En cuanto a las veces que entra en la zona central, la tendencia es similar siendo más acentuada en primer día del ensayo (Figura 2B). Estos resultados indican déficits conductuales en los ratones EMD similares a los observados en los modelos murinos de amiloidosis. A total of 62 male mice (27 EMD and 35 controls) were used in the study. On the other hand, the behavior of two lines of transgenic mice models of amyloidosis was studied, transgenic mice that overexpress the human APP695 peptide, and double transgenic mice APP / Ps1, a cross between mice that overexpress the human APP695 peptide and the Mutant form Ps1 (M146L). The behavior of the EMD mice and controls of 6 months of age was evaluated. In the T-labyrinth test, a significant increase in latency was found with respect to control mice that was similar to that shown by amyloidosis models, APP / PS1 mice and APP mice (Figure 2A). Regarding the open field test, again EMD mice behave similarly to amyloidosis models. The time remaining in the central area of the maze is significantly less, both in the first day as in 2 or 3 or and, in mice compared to controls. As for the times it enters the central zone, the trend is similar, being more pronounced on the first day of the trial (Figure 2B). These results indicate behavioral deficits in EMD mice similar to those observed in murine models of amyloidosis.
Finalmente la capacidad cognitiva fue estudiada mediante el test de memoria visual, sometiendo a los animales al test del reconocimiento de objetos. Los ratones EMD de 6 meses de edad pasan menos tiempo inspeccionando el objeto nuevo comparado con el tiempo que emplean los ratones controles (Figura 2C). Esta reducción en el tiempo que pasan con el objeto nuevo es similar al observado en los ratones APP/Ps1 y en los ratones APP (Figura 2C). Estos resultados indican que la memoria de trabajo está alterada en los ratones EMD de forma similar a lo sucedido en los modelos murinos de amiloidosis. Resumiendo, los estudios de comportamiento realizados a los ratones EMD a los 6 meses de edad reflejan un grado mayor de ansiedad. Por otra parte, estos animales también exhiben una significativa reducción en la capacidad de aprendizaje y la memoria espacial a corto plazo, la más afectada en la enfermedad de Alzheimer. Estos resultados están de acuerdo con un estudio in vivo reciente desarrollado sobre el efecto de la eliminación de la proteína LRP- 1 a nivel de la microvasculatura cerebral y la alteración en la memoria (Jaeger et al, 2009). En este estudio describen que la eliminación selectiva de la expresión de la proteína LRP-1 a nivel de la BBB reduce la capacidad de aprendizaje y memoria utilizando los mismos test de comportamiento (el laberinto en T y el reconocimiento de objetos). Con estos ejemplos se muestra que el modelo de ratón EMD desarrolla de forma natural y a una edad temprana (6 meses de edad) un fenotipo de comportamiento ansiolítico y de déficit de memoria semejante al que desarrollan otros modelos murinos de amiloidosis como son los ratones APP/Ps1 y los ratones APP. Finally, cognitive ability was studied using the visual memory test, subjecting the animals to the object recognition test. 6-month-old EMD mice spend less time inspecting the new object compared to the time spent by control mice (Figure 2C). This reduction in the time spent with the new object is similar to that observed in APP / Ps1 mice and in APP mice (Figure 2C). These results indicate that working memory is altered in EMD mice in a similar way to what happened in murine models of amyloidosis. In summary, behavioral studies performed on EMD mice at 6 months of age reflect a greater degree of anxiety. On the other hand, these animals also exhibit a significant reduction in learning capacity and short-term spatial memory, the most affected in Alzheimer's disease. These results are in agreement with a recent in vivo study developed on the effect of the elimination of LRP-1 protein at the level of cerebral microvasculature and memory impairment (Jaeger et al, 2009). In this study they describe that the selective elimination of LRP-1 protein expression at the BBB level reduces learning and memory capacity using the same behavioral tests (the T labyrinth and object recognition). With these examples it is shown that the EMD mouse model develops naturally and at an early age (6 months of age) a phenotype of anxiolytic behavior and memory deficit similar to that developed by other murine models of amyloidosis such as APP / mice. Ps1 and APP mice.

Claims

REIVINDICACIONES
1 . - Modelo animal no humano caracterizado por la ausencia de megalina producida mediante la deleción del gen LRP-2 localizada específicamente en el endotelio de los capilares cerebrales. one . - Non-human animal model characterized by the absence of megalin produced by the deletion of the LRP-2 gene located specifically in the endothelium of the cerebral capillaries.
2. - Modelo animal según la reivindicación 1 donde el animal no humano es un roedor o un primate. 2. - Animal model according to claim 1 wherein the non-human animal is a rodent or a primate.
3. - Modelo animal según la reivindicación 2 donde el roedor es un ratón o una rata. 3. - Animal model according to claim 2 wherein the rodent is a mouse or a rat.
4. - Modelo animal según cualquiera de las reivindicaciones 1 a 3 donde el animal es un animal transgénico. 4. - Animal model according to any of claims 1 to 3 wherein the animal is a transgenic animal.
5. - Procedimiento para la obtención de un modelo animal no humano según cualquiera de las reivindicaciones 1 a 4 que comprende la eliminación de megalina específicamente en el endotelio de los capilares cerebrales mediante la deleción del gen LRP-2. 5. - Method for obtaining a non-human animal model according to any of claims 1 to 4 comprising the removal of megalin specifically in the endothelium of the cerebral capillaries by deletion of the LRP-2 gene.
6. - Procedimiento según la reivindicación 5 donde la deleción del gen LRP-2 se produce mediante transgénesis. 6. - Method according to claim 5 wherein the deletion of the LRP-2 gene is produced by transgenesis.
7. - Procedimiento según la reivindicación 6 donde la deleción mediante transgénesis se realiza mediante el sistema Cre/lox. 7. - Method according to claim 6 wherein the deletion by transgenesis is performed by the Cre / lox system.
8. - Uso de un modelo animal según cualquiera de las reivindicaciones 1 a 4 para el estudio de una enfermedad neurodegenerativa. 8. - Use of an animal model according to any of claims 1 to 4 for the study of a neurodegenerative disease.
9. - Uso de un modelo animal según cualquiera de las reivindicaciones 1 a 4 para la identificación y/o evaluación de compuestos o composiciones para la prevención y/o el tratamiento de una enfermedad neurodegenerativa. 9. - Use of an animal model according to any of claims 1 to 4 for the identification and / or evaluation of compounds or compositions for the prevention and / or treatment of a neurodegenerative disease.
10. - Uso según cualquiera de las reivindicaciones 8 ó 9 donde la enfermedad neurodegenerativa es una enfermedad neurodegenerativa humana. 10. - Use according to any of claims 8 or 9 wherein the neurodegenerative disease is a human neurodegenerative disease.
1 1 . - Uso según cualquiera de las reivindicaciones 8 a 10 donde la enfermedad neurodegenerativa es la Enfermedad de Alzheimer. eleven . - Use according to any of claims 8 to 10 wherein the neurodegenerative disease is Alzheimer's disease.
PCT/ES2013/070693 2012-10-05 2013-10-07 Animal model with cognitive deficit, method for obtaining same, and applications WO2014053692A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ESP201231533 2012-10-05
ES201231533A ES2458921B1 (en) 2012-10-05 2012-10-05 ANIMAL COGNITIVE DEFICIT MODEL, OBTAINING PROCEDURE AND APPLICATIONS

Publications (1)

Publication Number Publication Date
WO2014053692A1 true WO2014053692A1 (en) 2014-04-10

Family

ID=50434399

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/ES2013/070693 WO2014053692A1 (en) 2012-10-05 2013-10-07 Animal model with cognitive deficit, method for obtaining same, and applications

Country Status (2)

Country Link
ES (1) ES2458921B1 (en)
WO (1) WO2014053692A1 (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007059242A1 (en) * 2005-11-15 2007-05-24 The University Of Rochester Low-density lipoprotein receptor-related protein 2 clears amyloid-beta peptide a cross the blood-brian barrier via apolipoprotein j

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007059242A1 (en) * 2005-11-15 2007-05-24 The University Of Rochester Low-density lipoprotein receptor-related protein 2 clears amyloid-beta peptide a cross the blood-brian barrier via apolipoprotein j

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BELL ROBERT D ET AL.: "Transport pathways for clearance of human Alzheimer's amyloid beta-peptide and apolipoproteins E and J in the mouse central nervous system", JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM : OFFICIAL JOURNAL OF THE INTERNATIONAL SOCIETY OF CEREBRAL BLOOD FLOW AND METABOLISM (2007), vol. 27, no. 5, 30 April 2007 (2007-04-30), pages 909 - 918 *
HAMMAD S M ET AL.: "Interaction of apolipoprotein J-amyloid beta-peptide complex with low density lipoprotein receptor-related protein-2/megalin. A mechanism to prevent pathological accumulation of amyloid beta-peptide..", THE JOURNAL OF BIOLOGICAL CHEMISTRY (997), vol. 272, no. 30, 25 July 1997 (1997-07-25), pages 18644 - 18649 *
HATCH E ET AL.: "Transgenic mouse models of A beta transport in Alzheimer's disease", 40TH ANNUAL MEETING OF THE SOCIETY-FOR-NEUROSCIENCE, vol. 40, 13 November 2010 (2010-11-13), SAN DIEGO, CA, USA *
OLIVEIRA ET AL.: "Genetic reduction of megalin increases brain Abeta plaque burden: Evidence from a new mouse model of Alzheimer's disease", 41ST ANNUAL MEETING OF THE SOCIETY-FOR-NEUROSCIENCE, vol. 41, 12 November 2011 (2011-11-12), WASHINGTON, DC, USA *
WILLNOW T ET AL.: "Defective forebrain development in mice lacking gp330/megalin", PROC. NATL. ACAD. SCI., vol. 93, 1996, pages 8460 - 8464 *
ZLOKOVIC B ET AL.: "Glycoprotein 330/megalin: Probable role in receptor-mediatedtransport of apolipoprotein J alone and in a complex with Alzheimer disease amyloid 13 at the blood-brain and blood-cerebrospinal fluid barriers", PROC. NATL. ACAD. SCI., vol. 93, 1996, pages 4229 - 4234 *

Also Published As

Publication number Publication date
ES2458921B1 (en) 2015-04-01
ES2458921A1 (en) 2014-05-07

Similar Documents

Publication Publication Date Title
de Pins et al. Conditional BDNF delivery from astrocytes rescues memory deficits, spine density, and synaptic properties in the 5xFAD mouse model of Alzheimer disease
Blumenstock et al. Seeding and transgenic overexpression of alpha‐synuclein triggers dendritic spine pathology in the neocortex
Royle et al. Behavioural analysis and susceptibility to CNS injury of four inbred strains of mice
Majid et al. In vivo axonal transport deficits in a mouse model of fronto-temporal dementia
Sager et al. Transgenic zebrafish models of neurodegenerative diseases
Hansen et al. A novel α-synuclein-GFP mouse model displays progressive motor impairment, olfactory dysfunction and accumulation of α-synuclein-GFP
Snyder et al. Nervous system
Sennvik et al. Tau‐4R suppresses proliferation and promotes neuronal differentiation in the hippocampus of tau knockin/knockout mice
Yin et al. New pathogenic insights from large animal models of neurodegenerative diseases
Osking et al. ALS-linked SOD1 mutants enhance neurite outgrowth and branching in adult motor neurons
de Rus Jacquet et al. Current and future applications of induced pluripotent stem cell-based models to study pathological proteins in neurodegenerative disorders
Neveklovska et al. Deletion of the huntingtin proline-rich region does not significantly affect normal huntingtin function in mice
Lim et al. Galectin-3 secreted by human umbilical cord blood-derived mesenchymal stem cells reduces aberrant tau phosphorylation in an Alzheimer disease model
Sharkey et al. Modeling UBQLN2-mediated neurodegenerative disease in mice: Shared and divergent properties of wild type and mutant UBQLN2 in phase separation, subcellular localization, altered proteostasis pathways, and selective cytotoxicity
Pu et al. Generation of Alzheimer’s disease transgenic zebrafish expressing human app mutation under control of zebrafish appb promotor
Jiang et al. Amyloid precursor protein intracellular domain-dependent regulation of FOXO3a inhibits adult hippocampal neurogenesis
ES2830173T3 (en) Non-human animal with lower and upper motor neuron function and decreased sensory perception
Cloyd et al. Effects of altered tau expression on dentate granule cell excitability in mice
ES2670590T3 (en) Upper corticospinal motor neurons, methods and compositions for differentiating neural stem cells by modulating CB1 cannabinoid receptor signaling and uses thereof
Plucińska et al. Imaging of neuronal mitochondria in situ
Guha et al. ICAM-1 protects neurons against Amyloid-β and improves cognitive behaviors in 5xFAD mice by inhibiting NF-Κb
WO2014053692A1 (en) Animal model with cognitive deficit, method for obtaining same, and applications
Mi et al. Mutation of a ubiquitin carboxy terminal hydrolase L1 lipid binding site alleviates cell death, axonal injury, and behavioral deficits after traumatic brain injury in mice
Fuentealba Understanding the role of perivascular macrophages in Parkinson's disease pathophysiology
Jang et al. RAPGEF2 mediates oligomeric Aβ-induced synaptic loss and cognitive dysfunction in Alzheimer’s disease

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13843908

Country of ref document: EP

Kind code of ref document: A1

DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13843908

Country of ref document: EP

Kind code of ref document: A1