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WO2010037884A1 - Composition cellulaire in vitro à surexpression simultanée de gènes humains mutés ps1 m146v, app swe et tau p301l - Google Patents

Composition cellulaire in vitro à surexpression simultanée de gènes humains mutés ps1 m146v, app swe et tau p301l Download PDF

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Publication number
WO2010037884A1
WO2010037884A1 PCT/ES2009/070304 ES2009070304W WO2010037884A1 WO 2010037884 A1 WO2010037884 A1 WO 2010037884A1 ES 2009070304 W ES2009070304 W ES 2009070304W WO 2010037884 A1 WO2010037884 A1 WO 2010037884A1
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WIPO (PCT)
Prior art keywords
tau
amyloid
disease
beta
app
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Ceased
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PCT/ES2009/070304
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English (en)
Spanish (es)
Inventor
Luis M. BOTANA LÓPEZ
Lydia GIMÉNEZ- LLORT
María del Carmen VALE GONZÁLEZ
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universidade de Santiago de Compostela
Universitat Autonoma de Barcelona UAB
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Universidade de Santiago de Compostela
Universitat Autonoma de Barcelona UAB
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Publication of WO2010037884A1 publication Critical patent/WO2010037884A1/fr
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Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals

Definitions

  • the present invention relates to an in vitro model obtained from 3xTg-AD mice for Alzheimer's disease with simultaneous overexpression of PS1 MU ⁇ V, APP S we and tau P3on as well as its usefulness for the study of the biological basis of the disease Alzheimer's disease and / or diseases related to tau and beta-amyloid pathology and for the evaluation of preventive, therapeutic and / or palliative therapies for this disease.
  • AD Alzheimer's disease
  • AD Alzheimer's disease
  • the data available in the Alzheimer Spain Foundation http://www.fundacionalzheimeresp.org
  • about 700,000 people are affected in Spain and more than 100,000 new patients are manifested a year, which represents more than two million Vietnamese Vietnamese Vietnamese condominiums and his relatives, today, see his life upset by the disease.
  • 8 million Europeans are affected by Alzheimer's disease and also taking into account the aging of the population, the number of patients is expected to double in 2020 and triple in 2050.
  • AD Alzheimer's disease
  • amyloid deposits are extracellular fibers formed by ⁇ -amyloid, ( ⁇ A, specifically ⁇ A40 and ⁇ A42), peptides generated by the proteolytic breakdown of the ⁇ -amyloid precursor protein (APP).
  • Neurofibrillar tangles in contrast, are intracellular filaments formed by the polymeration of tau, which normally functions as a protein associated with microtubules in neuronal axons.
  • APP beta-amyloid precursor protein
  • beta-amyloid peptide In the brains of patients with AD, amyloid plaques containing aggregates of beta-amyloid appear in specific brain regions, triggering an inflammatory response, neuronal death and progressive cognitive impairment.
  • One of the mechanisms by which the beta-amyloid peptide is generated from APP is the rupture of APP in an extracellular position (beta site) followed by an unusual rupture within the transmembrane segment of APP (gamma site), generating a fragment APP containing 39-43 amino acids.
  • beta site extracellular position
  • gamma site gamma site
  • Several mutations in the APP protein have been correlated with the EA due to the increase or alteration in the transformation of APP into beta-amyloid, in particular the transformation of APP to large amounts of the long form of beta-amyloid (i.e.
  • beta-amyloid 40 and beta-amyloid 42 The ⁇ -amyloid peptide has also been implicated in neuropathological defects in individuals with Down syndrome. For example, practically all individuals with Down syndrome, who have an extra copy of chromosome 21 show neuropathological alterations similar to those observed in Alzheimer's disease if they survive beyond 40 years and this seems to be due to an excessive production of ⁇ -amyloid, which is encoded by the APP gene located on chromosome 21.
  • Alzheimer's disease In recent years, animal models for Alzheimer's disease have provided excellent opportunities to deepen knowledge of the pathophysiology and therapies for the disease.
  • the main characteristics of Alzheimer's disease have been reproduced in different murine models obtained from transgenic mice, obtained by genetic engineering incorporating some of the transgenes that in humans are present in the few cases (only 10%) of type Alzheimer family, mainly models that overexpress the APP gene that gives rise to the ⁇ A precursor protein.
  • none of these models had managed to combine the main neuropathological, behavioral and neurochemical characteristics of Alzheimer's disease until in 2003 Professor LaFerla developed the 3xTgAD triple transgenic mice.
  • 3xTgAD mice with human transgenes PS1 MU ⁇ V, APP Swe and taup30fL, present the most outstanding characters of EA and progressively develop ⁇ A plaques and neurofibrillar tangles of Tau in a temporal and neuroanatomic pattern parallel to that of humans .
  • they present synaptic dysfunction, cholinergic and behavioral deficiencies, depending on age but starting before the pathology of plaques and tangles, and long-term synaptic plasticity deficits correlate with the accumulation of ⁇ A at the intracellular level.
  • 3xTg-AD mice provide a unique animal model to study the neurobiology of the disease and evaluate potential preventive and therapeutic strategies on both types of lesion (tau and beta-amyloid pathology) and other cellular and molecular deficiencies present in Alzheimer's disease.
  • AD Alzheimer's disease
  • a first aspect of the present invention relates to an in vitro cellular model to evaluate the efficacy of compounds and materials to modify, treat or prevent diseases related to tau, beta-amyloid or APP pathology that includes the use of primary cultures of cortical neurons obtainable from embryos of 3xTgAD mice grown under depolarizing conditions.
  • Pulthologies related to tau, beta-amyloid or APP pathology means any disease that occurs with an increase in aggregates of tau protein, and / or increased levels of beta-amyloid or beta-amyloid precursor protein.
  • the compounds evaluated are selected from the group consisting of peptides, polypeptides, proteins, biological agents or drugs.
  • the disease is Alzheimer's disease.
  • the evaluation is carried out using HTS (high through-put screening) methods.
  • primary neuron cultures are obtained from embryos of 3XTgAD mice whose age is between 15-17 days.
  • a second aspect of the present invention refers to a composition (hereinafter composition of the present invention) comprising primary cultures of cortical neurons obtainable from the cerebral cortex of embryos of 3xTgAD mice of 15-17 days gestation and that remain in depolarizing conditions.
  • the "3xTgAD mice” described in the present invention refer to triple transgenic mice obtainable by the procedure detailed in the international application WO2003 / 053136 and provided by the holders of said application.
  • a third aspect of the present invention relates to a method of preparing the composition of the invention.
  • a fourth aspect of the present invention refers to the use of the composition of the invention for the study of the biological bases of diseases related to tau, beta-amyloid or APP pathology.
  • the composition of the invention is used to evaluate the efficacy of compounds and materials to modify, treat or prevent diseases related to tau, beta-amyloid or APP pathology.
  • the composition of the disease is Alzheimer's disease.
  • Figure 1 shows an image of confocal microscopy in which the expression of beta-amyloid precursor protein (APP) in wild cultures (A) and neocortical cultures obtained from triple transgenic animals (B) is represented. At 8 days in culture the expression of APP is increased in primary cultures obtained from triple-transgenic animals. 20 ⁇ m scale bar.
  • APP beta-amyloid precursor protein
  • Figure 2 shows an image of confocal microscopy in which the expression of beta-amyloid in wild cultures (A) and neocortical cultures obtained from triple transgenic animals (B) is represented. At 8 days in culture the expression of ⁇ A is increased in primary cultures obtained from triple-transgenic animals. 20 ⁇ m scale bar.
  • Figure 3 shows an image of confocal microscopy in which the expression of Tau 46 (total tau) in wild cultures (A) and neocortical cultures obtained from triple transgenic animals (B) after 8 div. At 8 days in culture the expression of total Tau is increased in primary cultures obtained from triple-transgenic animals. 20 ⁇ m scale bar.
  • Figure 4 shows an image of confocal microscopy in which the expression of Tau HT7 (residues 159-163 of the protein) in wild cultures (A) and neocortical cultures obtained from triple transgenic animals (B) after 8 div. After 8 days in culture, the expression of Tau is increased in primary cultures obtained from triple-transgenic animals. 20 ⁇ m scale bar.
  • Figure 5 shows an image of confocal microscopy in which the expression of Tau AT8 (phosphorylated Tau in Ser 202) is represented in wild cultures (A) and neocortical cultures obtained from transgenic triple animals (B) after 8 div. At 8 days in culture the expression of phosphorylated Tau in Ser 202 is increased in primary cultures obtained from triple-transgenic animals. 20 ⁇ m scale bar.
  • Figure 6 shows an image of confocal microscopy in which the expression of Tau AT100 (phosphorized tau in Thr 212 and Ser 214) is represented in wild cultures (A) and neocortical cultures obtained from transgenic triple animals (B) after 8 div . At 8 days in culture the expression of this protein isoform is increased in primary cultures obtained from triple-transgenic animals. 20 ⁇ m scale bar.
  • Figure 7 shows an image of confocal microscopy in which the expression of Tau AT270 (Tau phosphorilated in Thr 181) is represented in wild cultures (A) and neocortical cultures obtained from transgenic triple animals (B) after 8 div. At 8 days in culture the expression of this protein isoform is increased in primary cultures obtained from triple-transgenic animals. 20 ⁇ m scale bar.
  • Figure 8 shows an image of confocal microscopy in which the expression of Tau 46 (total tau) in wild cultures (A) and neocortical cultures obtained from embryos of triple transgenic animals (B) after 14 div. At 14 days in culture the expression of total Tau is increased in primary cultures obtained from triple-transgenic animals. 20 ⁇ m scale bar.
  • the present invention faces the technical problem of finding a solution that solves the problems associated with the in vivo transgenic models for AD that exist today.
  • the present invention provides a new, fast, efficient and reproducible in vitro model to study the biological bases and evaluate therapies and compounds for the treatment and prevention of Alzheimer's disease and / or diseases associated with Tau and ⁇ A based on the use of a composition comprising primary cultures of cortical neurons obtainable from embryos of 3xTgAD mice that are maintained in vitro under depolarizing conditions.
  • the human transgenes PS1 MU ⁇ V , APPswe and taup30 ⁇ /. are expressed under the control of the Thy1.2 promoter that it directs their expression predominantly to the central nervous system, and they are already expressed in embryonic stages.
  • human transgenes are activated in neuronal cultures in vitro as demonstrated in the present invention and are also overexpressed in a time-dependent manner in culture, therefore the Primary neuronal cultures obtained from embryos of 3xTgAD mice constitute a unique model for AD.
  • the indispensable culture conditions to achieve a time-dependent gene expression are depolarizing conditions (10-25 mM KCI) and high seeding density (more than 2,800,000 cells per ml).
  • this in vitro model provides a unique multidimensional model to study the biological basis of the disease and evaluate preventive and / or therapeutic strategies for AD since it has been shown that some treatments in models that overexpress only beta-amyloid or tau can improve one of the pathologies and worsen the other (for example chronic nicotine treatments improve ⁇ -amyloid deposits but worsen tangles neurofibrillary). To date, there is no in vitro model that reproduces at a cellular, molecular and functional level the neuropathological spectrum of Alzheimer's disease that human patients show.
  • the proposed invention describes an in vitro model to study the biological basis of EA and evaluate the efficacy of compounds and materials, such as biological products, drugs and chemical products or neurochemical interactions resulting from cultures obtained from hybrid animals, in modify, treat or prevent Alzheimer's disease and other diseases related to tau, beta-amyloid or APP pathology.
  • the method involves the use of primary cultures of cortical neurons obtained from an animal model for Alzheimer's disease, 3xTgAD triple transgenic mice.
  • Cell cultures are obtained from fetuses of 3xTgAD mice and compared to untreated cultures or cultures obtained from non-3xTgAD control mice thus providing an in vitro system to evaluate the effects of different compounds on the biochemical and functional properties of neurons. control and neurons with neuropathology characteristic of AD and / or related diseases.
  • This method provides an economic, reliable and fast model and allows the use of analysis techniques that do not require waiting to reach a certain state of development of the animal as it is used in in vivo models for Alzheimer's disease.
  • primary cell cultures preferably cortical cultures, obtained from 3xTgAD triple transgenic animals
  • 3xTgAD mice have numerous advantages over other preparations such as primary neuronal cultures obtained from transgenic mice for AD that overexpress only beta-amyloid or tau, brain slices or experiments in vivo to evaluate the efficacy of compounds in the treatment or prevention of Alzheimer's disease.
  • the proposed invention describes several utilities based on the discovery that this model has overexpression of ⁇ -amyloid, APP and Tau.
  • Example 1 Obtaining primary neuronal cultures
  • primary neuronal cultures obtained from fetuses of 15-17 days of gestation of 3xTgAD transgenic triple animals constitute a cellular model with simultaneous overexpression of APP, tau and Beta-amyloid Io that allows the in vitro study of the bases Biological aspects of Alzheimer's disease, including gene and protein expression, enzymatic activities, neurotransmission systems, functional properties of neurons affected by AD and of the elements involved in the beta-amyloid cascade as well as inflammatory and excitotoxic or oxidative stress associated with the pathology of AD.
  • primary cultures are established from embryos of 15-17 days of gestation obtained from 3xTgAD mice and non-3xTgAD control mice.
  • the procedure used to obtain primary cortical cultures according to the present invention is as follows:
  • Cell cultures are obtained from the cerebral cortex of embryos of 3xTgAD and non-3xTgAD mice between 15 and 17 days of gestation.
  • Pregnant females are sacrificed by anesthesia with ether, the abdomen is disinfected with 70% ethanol and the fetuses are removed.
  • the uterus, containing the fetuses is transferred to a laminar flow hood in a Petri dish and then the fetuses are removed.
  • the cerebral cortex is separated from the rest of the brain and the cells are dissected using enzymatic treatment and mechanical disintegration.
  • the fetal cerebral cortex is removed, cut and dissociated by trypsin treatment and subsequent treatment with solution containing trypsin and DNAse inhibitor.
  • Dissociated cells are centrifuged (5 minutes at 1000 rpm) and resuspended in Dulbecco culture medium containing 10% v / v fetal bovine serum, 10-25 mM KCI, 0.2 mM glutamine, 10 units / l penicillin G , 0.1 IU / I insulin and 7.3 ⁇ M of p-aminobenzoic acid.
  • the cells are seeded in culture plates pretreated with poly-D-lysine (between 10 and 60 mg / l) and kept at 37 ° C in a humid atmosphere with 5% CO2 / 95% air.
  • cytosine arabinoside (20 ⁇ M) is added to prevent the proliferation of non-neuronal cells in the event that it is desired to obtain cultures of pure cortical neurons. This last step can be avoided when it is necessary to obtain mixed cultures of neurons and glia. From 6 days in culture, the overexpression of APP, tau and beta-amyloid has been characterized in primary neuronal cultures obtained from 3xTgAD mice compared to primary neuronal cultures obtained from wild mice subjected to the same procedure (non-3xTgAD ).
  • Example 2 Evaluation of the efficacy of compounds and materials using the composition of the present invention.
  • primary neuronal cultures of 3xTg-AD triple transgenic animals for Alzheimer's disease have been characterized to evaluate the efficacy of compounds and materials (eg, drugs and other biological and synthetic agents and substances) in the treatment or prevention of Alzheimer's disease or diseases associated with beta-amyloid and / or tau and APP.
  • Compounds that can be evaluated in this model include small molecules such as peptides and proteins, biological agents, chemical compounds and drugs. Preferably, they will be non-toxic compounds and well tolerated to be used in the treatment, therapy and prevention of AD or diseases associated with beta-amyloid and / or tau.
  • the responses of the cells in this cellular model can be determined quantitatively, thus allowing to determine the activity or effect of a compound in the molecular, biochemical and physiological mechanisms that take place in the cell and that are associated with AD or diseases. related to beta-amyloid and / or tau.
  • neuronal cultures obtained from 3xTgAD and non-3xTgAD animals are fixed in 4% paraformaldehyde and subsequently labeled with primary antibodies against APP / ⁇ A (NAB228: 1: 50), ⁇ -amyloid 6E10 (1: 500), Tau 46 (Total Tau 1: 500), Tau HT7 (1: 200, residues 159-163), Tau AT8 (Tau phosphorylated at Ser 202, 1: 100), Tau AT100 (1: 100, tau phosphorilada in Thr 212 and Ser 214) and Tau AT270 (1: 100, Tau phosphorilada in Thr 181).
  • Immunoreactivity is visualized using a secondary fluorescent antibody in confocal microscope (Nikon, Mellville, NY 1 USA), with a Hamamatsu ORCA-ER camera (Hamamatsu Photonics KK, Hamamatsu, Japan).
  • the controls for the specificity of the detection methods were performed by omitting the incubation with the primary antibody in one of every four consecutive wells. None of the wells without primary antibody showed no signal comparable to the signal obtained with the primary antibodies.
  • This process can be automated for HTS (high throughput screening) using culture plates to which cells were previously added, which were cultured for at least 6 days. These plates are susceptible of being incorporated into automatic systems of measurement of the markers of interest, by different measurement methods (absorbance, luminescence, fluorescence).
  • HTS high throughput screening
  • these plates are susceptible of being incorporated into automatic systems of measurement of the markers of interest, by different measurement methods (absorbance, luminescence, fluorescence).
  • the effect of different therapies on cellular, molecular and biochemical targets of relevance for Alzheimer's disease is likely to be investigated in this model in vitro by means of HTS methods using commercial kits of interest for the evaluation of the effect of a treatment of any nature in the evolution of AD or diseases associated with tau and beta-amyloid, after the cells have undergone the treatment in question.

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Abstract

L'invention concerne une composition qui comprend des cultures primaires de neurones corticaux pouvant être obtenues à partir d'embryons de souris 3XTgAD cultivés dans des conditions dépolarisantes. L'invention concerne également une méthode in vitro pour évaluer l'efficacité de composés et de matériaux pour modifier, traiter ou prévenir des maladies associées à une pathologie tau, bêta-amyloïde ou APP qui consiste à utiliser des cultures primaires de neurones corticaux pouvant être obtenues à partir d'embryons de souris 3XTgAD cultivés dans des conditions dépolarisantes.
PCT/ES2009/070304 2008-10-02 2009-07-23 Composition cellulaire in vitro à surexpression simultanée de gènes humains mutés ps1 m146v, app swe et tau p301l Ceased WO2010037884A1 (fr)

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ES200802799A ES2335848B1 (es) 2008-10-02 2008-10-02 Composicion celular in vitro con sobreexpresion simultanea de genes humanos mutados ps1 m146v, app swe y tau p301l.
ESP200802799 2008-10-02

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003053136A2 (fr) * 2001-12-20 2003-07-03 The Regents Of The University Of California Modele de souris triplement transgenique de la maladie d'alzheimer

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003053136A2 (fr) * 2001-12-20 2003-07-03 The Regents Of The University Of California Modele de souris triplement transgenique de la maladie d'alzheimer

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FAURE, J. ET AL.: "Exosomes are released by cultured cortical neurones.", MOLECULAR AND CELLULAR NEUROSCIENCES., vol. 31, no. 4, April 2006 (2006-04-01), pages 642 - 648 *
HASEGAWA, T. ET AL.: "Etiological compound of homocysteic acid for Alzheimer's disease and preventive and cure effect of pyroloquinoline quinone (PQQ)", ALZHEIMER'S & DEMENTIA: THE JOURNAL OF THE ALZHEIMER'S ASSOCIATION, vol. 3, no. 3, 1 July 2007 (2007-07-01), pages S122 *
ROMANO, A. ET AL.: "Neuronal fibrillogenesis: amyloid fibrils from primary neuronal cultures impair long-term memory in the crab Chasmagnathus.", BEHAVIOURAL BRAIN RESEARCH., vol. 147, no. 1-2, 17 December 2003 (2003-12-17), pages 73 - 82 *

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ES2335848A1 (es) 2010-04-05

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