RU2532525C2 - Exogenic-induced animal model of alzheimer disease - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to experimental medicine and concerns modelling Alzheimer disease. That is ensured by using B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J oncomice. A preparation containing a synthetic analogue of aspartic acid isomerised in an amino acid residue in position 7 of human beta-amyloid with the amino acid sequence DAEFRH[isoD]SGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA) and/or fragments thereof containing the isomerised aspartic acid residue in position 7 [isoD] into the animal's blood-vascular system. The preparations are administered in a dose of 100 mcg, in 200 mcl/head of the solution once a month.

EFFECT: method enables a spontaneous change of a rate of the pathological process progression in the test animals depending on the purposes of the specific study by varying the number of injections and/or an amount of synthetic peptide per one injectable dose.

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Description

The technical field to which the invention relates.

The invention relates to a method for creating a model of Alzheimer's disease, characterized in that cerebral amyloidosis in experimental animals is caused by the introduction into their body of synthetic analogues of aspartic acid isomerized by the amino acid residue at position 7 of human amyloid beta and / or its fragments, including the isomerized aspartic acid residue in position 7.

State of the art

Alzheimer's disease (AD) is a fatal neurodegenerative pathology, the clinical course of which is accompanied by a steady decline in the patient's psychomotor functions over a long period (1). In Russia, the number of such patients is about one and a half million people (2). Medicines that can stop the course of this pathology currently do not exist anywhere in the world, but their search is given enormous importance in all developed countries, where the number of people suffering from Alzheimer's disease increases with an increase in the number of elderly people.

A characteristic molecular process of Alzheimer's disease is the conformational conversion of a small (39-43 amino acid residue) protein, amyloid beta (3). This protein is a normal component of the blood, where it is present in the form of a monomer, however, it forms oligomers and supramolecular aggregates (amyloid plaques) in patients with clinically diagnosed Alzheimer's disease (4). According to the widely accepted amyloid hypothesis, it is the polymerization of amyloid beta, which leads to cerebral amyloidosis, that is the key event that triggers the entire pathogenic cascade of Alzheimer's disease (5).

Cerebral amyloidosis is the formation of dense congophilic amyloid plaques in specific sections of the brain and is one of the most important neuromorphological signs of Alzheimer's disease (6). Currently, there are several animal models of Alzheimer's disease based on the use of transgenic rodents (mice, rats). In these models, cerebral amyloidosis is caused by changes in the genome that lead to increased expression of endogenous human beta-amyloid (Aβ) in the blood (7-9). Mice and rats of the wild type, unlike all other mammals, have three substitutions in the amino acid sequence of beta-amyloid and are not susceptible to Alzheimer's disease. The introduction of synthetic Aβ analogues into mammals does not cause them to develop cerebral amyloidosis and, accordingly, the pathogenesis of AD. At the same time, it was shown that intracerebral injections of homogenized amyloid plaques isolated from the brain of people affected by Alzheimer's disease led to the development of cerebral amyloidosis in experimental animals (10-19). These data suggested that the main driving force behind AD pathogenesis is the aggregation of endogenous amyloid beta under the influence of structurally and / or chemically altered amyloid beta isoform, which is contained in extracts of amyloid plaques (20-21). Nevertheless, despite numerous studies over more than 20 years, no scientific group in the world has been able to find such an isoform of amyloid beta. Accordingly, at the time of the creation of the present invention, nothing was known about the precise identification of an Alzheimer's disease-inducing agent, and moreover, no one was able to create an exogenously-induced animal model of Alzheimer's disease

In 2008, the authors of this invention showed that isomerization of the aspartic acid residue at position 7 leads to zinc-dependent oligomerization of the metal-binding domain of amyloid beta (22). Since amyloid plaques contain up to 75% of amyloid beta isomerized in this way and an excess of zinc ions, we, the first and only in the world, suggested that it is the zinc complexes of this beta amyloid isoform that can be the molecular agent causing oligomerization and subsequent aggregation soluble forms of beta-amyloid, that is, those same processes that the vast majority of researchers consider the triggers of the pathogenesis of Alzheimer's disease. It is important to note that in the work of M. Meyer-Luehmann et al (14) and in all other known works of our time, the ability of our agent to cause cerebral amyloidosis has not been tested. Thus, by the time the present invention was created, no one was able to justifiably indicate which isoform of beta-amyloid is the initiator of cerebral amyloidosis, which certainly indicates the complete non-obviousness of the present invention.

SUMMARY OF THE INVENTION

The present invention consists in the exogenous initiation of cerebral amyloidosis in mammals that express endogenous human beta-amyloid in physiologically determined or artificially high amounts. In the present invention, for the first time in the world, injections of preparations containing synthetic analogues of aspartic acid isomerized by the amino acid residue of aspartic acid at position 7 of human beta-amyloid (isoAsp7-beta-amyloid) and / or its fragments, including the balance of isomerized aspartic acid at position 7. The advantage of such exogenously-induced models of Alzheimer's disease in comparison with currently existing constit tional transgenic models is the possibility to arbitrary change of the speed of pathological processes in experimental animals according to the specific objectives of the study. An additional advantage of this invention is the possibility of using non-asgenic animals as a model of Alzheimer's disease, as it is obvious for specialists in this field that if a molecular agent causes cerebral amyloidosis in transgenic mice expressing human amyloid beta, then this same agent, namely preparations containing synthetic analogs of isomerized aspartic acid residue at position 7 of human amyloid beta and / or its fragments, including yuchayuschih isomerized residue aspartic acid at position 7 - will inevitably cause cerebral amyloidosis all other mammals in which the human beta-amyloid present constitutionally.

The technical result.

The technical result achieved by using the patented invention is to create an exogenously-induced animal model of Alzheimer's disease. No prototypes of this invention exist anywhere in the world, since only within the framework of the present invention the role of preparations containing synthetic analogues of aspartic acid isomerized at the amino acid residue at position 7 of human amyloid beta and / or its fragments, including the isomerized residue, is shown for the first time. aspartic acid at position 7, as exogenously administered agents inducing the pathology of Alzheimer's disease.

An example embodiment of the invention.

To create an exogenously inducible model of Alzheimer's disease, we used the line of transgenic mice B6C3-Tg (APPswe, PSEN1dE9) 85Dbo / J (JAX® GEMM® B6C3-Tg (APPswe, PSEN1dE9) 85Dbo / J) without a specific pathogenic microflora. The mice of this strain have the following changes in the genome (21):

- The human gene encoding the mutated form ("Swedish version", APPswe, K670N / M671L) Amyloid beta precursor protein; this form causes hereditary Alzheimer's disease.

- The human gene encoding the mutated form (A246E) of Presenelin 1; this form also causes hereditary Alzheimer's disease.

Both genes are under the control of a mouse prion protein promoter. The transgenic product was introduced into the fertilized eggs of C57BL / 6JXC3HeJF2 mice; the successful line was isolated and propagated by crossbreeding with C57BL / 6J mice for 14 generations.

B6C3-Tg mice (APPswe, PSEN1dE9) 85Dbo / J are characterized by neuropathological lesions that closely resemble those found in patients with Alzheimer's disease (amyloid plaques, neurofibrillary tangles, neuronal death, psychomotor disturbances ...) and are commercially available.

For the period of the experiments, transgenic mice were kept in sterile conditions (Pushchino, a branch of the Institute of Organic Chemistry, Russian Academy of Sciences). All work with animals was carried out using personal protective equipment (technological clothing). All consumables used in the experiments (syringes, ampoules, gauze, cotton wool), as well as the bodies of dead and euthanized animals, were subjected to specialized accumulation and further disposal at the fire waste disposal station of the FIBCH RAS. For the accumulation of used needles, a Sharpe container was used. During the experiment, the standard conditions for keeping animals were used in accordance with the Program for the care and maintenance of animals in the PJ, reviewed and approved by the Institute Commission for the Control of the Use and Use of Laboratory Animals in July 2009.

As injectables were used;

- "A": a solution of beta-amyloid in water (concentration = 100 μm)

- “B”: Solution of isoAsp7-run-amyloid in water (concentration = 100 μM)

"Aβ42" (active component of the drug "A"): synthetic 42-membered peptide DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA. The amino acid sequence of this peptide corresponds to human beta-amyloid (Beta-amyloid protein 42, or Aβ42), which is a fragment 672-713 of the Amyloid precursor protein (Amyloid beta A4 protein, UniProtKB / Swiss-Prot P05067, A4_HUMAN). Average molecular weight: 4514 g / mol. "[IsoD7] -Aβ42" (active ingredient of the drug "B"): synthetic 42-membered peptide DAEFRH [isoD] SGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA. This peptide is an [isoD7] analogue of the substance Aβ42. Average molecular weight: 4514 g / mol.

All substances and preparations used are low-hazardous chemicals (4th hazard class).

Given that the average blood volume in a mouse is 2 ml, and the concentration of amyloid beta in the blood of a transgenic Alzheimer's mouse is 200 nM, the total circulating beta amyloid content in this animal is about 0.002 · 200 · 10-9 = 0.4 nanomol = 2000 ng (2 mcg). In accordance with the purpose of research, the amount of the drug administered should be at least 25 times higher than the amount of native amyloid beta and, accordingly, should be 25 * 2000 ng = 50,000 ng (50 μg). Accordingly, the concentration of isoform of beta-amyloid in 100 μl of the injected solution of the drug can be calculated from the ratio:

25 · 0.200 μm · 2000 μl = x μM · 100 μl,

and will be 100 μM. Moreover, in 100 μl of the injected drug, the total amount of beta-amyloid will be 50 (fifty) μg, and in 200 μl - 100 (one hundred) μg.

The age of the animal to the first injection = 8-10 weeks. Each drug was injected once a month intravenously into the retroorbital venous plexus in a volume of 200 μl / goal, a total of 8 injections were performed for each animal, the last injection was performed at the age of 9 months.

Intravenous injection into the retroorbital venous plexus in mice is permitted for administration in the absence of anesthesia. To carry out this procedure, the mouse is grabbed by the thumb and forefinger on the skin of the neck, and is firmly held by the other fingers on the skin of the back and pressed to the content grid. The conjunctiva of the inner corner of the eye is pierced with a syringe needle and held to a depth of 1-2 mm for the eyeball, where the venous plexus is located. When correctly inserted into the needle from the retroorbital plexus, blood flows by gravity (if in doubt, you can create a small negative pressure in the syringe). After ascertaining the correct location, the test drug is slowly introduced. After injection with a sterile gauze, the eyeball is slightly pressed to stop the bleeding. In this way, the drug can be administered with a 1 ml syringe and a No. 27-29G½ needle. The volume of the injected drug is 100-200 μl / goal. Throughout the experiment, the mice are individually kept in Type-2 cells with identification plates that indicate the number of animals, the name of the line, gender, type and date of manipulation. Anesthesia will be used to relieve pain and stress during intravenous injection into the retroorbital venous plexus. The main pain and stress are associated with fixation and intravenous injection. At the end of the experiment, all animals were euthanized in each group and preparations of the brain were prepared. Euthanasia was carried out in accordance with the Russian national sanitary legislation and the American legislation on the protection of animals with carbon dioxide in accordance with the Protocol-application for manipulating animals No. 09/01 approved by the IACUC Commission.

Morphological analysis of brain tissue of transgenic animals was carried out using histochemical methods described below.

Coating glass slides with gelatin and alum-chrome alum - 2 g of gelatin was dissolved in 400 ml of distilled water, with the solution heated to 55 ° C with constant stirring. After dissolution, 0.2 g of alum-chrome alum was added and the solution was well mixed. The solution was heated to 60 ° C to cover the slides. Glasses were dried during the day before use.

Brain Fixation - The mouse brain is removed from the cranium and placed in a freshly prepared solution of 4% paraformaldehyde in phosphate buffer (pH 7.4) for six days with a three-time change of solution after two days. On the seventh day, immediately before the slicing, the brain is placed for six days in a 30% sucrose solution with a three-time change of solution after two days.

Slicing - The mouse brain is removed from the sucrose solution, blotted with filter paper and covered with a freezing medium (e.g. Tissue-Tek® OST Compound, Thermo). Further, the brain was frozen either on a Peltier element, which is equipped with a Thermo cryote, or in liquid nitrogen. After freezing, the brain was placed in a cryotome and sections were made 30 microns thick. Slices were placed on a glass slide coated with gelatin and histologically stained.

Histological staining

(1) Mayer staining of hematoxyliomas: Sections were dehydrated successively for 2 minutes in 100%, 96% and 70% alcohol and placed in water for 2 minutes. Then the preparations are placed for 30 min in a hematoxylin solution, washed with water and developed in TAP water until a blue color appears.

(2) Staining of amyloid plaques with Congo Red alcohol solution; Sections previously dehydrated in alcohol solutions and stained with Mayer hematoxylin are incubated in a saturated 80% alcohol solution of NaCl containing 1% of one percent NaOH for 20 minutes. Then the drug is placed in a solution of Congo red containing 1% of one percent NaOH for 20 minutes. The preparations are dehydrated twice in 5 minutes in 100% alcohol, enlightened in xylene and enclosed in a balm. The visualization of pink amyloid plaques is carried out in transmitted light and confirmed in polarization. For the simultaneous staining of amyloid and cellular structures, an approach was applied with staining of cells with heier hematoxylin according to Mayer and plaques Congo-red.

The number of congophilic amyloid plaques was calculated manually using a light microscope in polarized light. The results of the comparative analysis showed (Table 1, Figure 1) that the number of plaques in mice injected with the synthetic analogue of human amyloid beta isomerized aspartate-7 synthetic analogue significantly exceeds the number of plaques in control animals injected with the preparation containing intact amyloid beta, which unequivocally testifies to the ability of human amyloid beta-isomerized aspartate-7 to cause cerebral amyloidosis in appropriately treated animals.

Claims (1)

A method of creating a model of Alzheimer's disease, which consists in introducing into the circulatory system of the B6C3-Tg (APPswe, PSEN1dE9) 85Dbo / J line of transgenic mice a preparation containing a synthetic analogue of aspartic acid isomerized by the amino acid residue at position 7 of the human amyloid beta at the amino acid sequence DAEFRH [isoD] SGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA) and / or a fragment thereof comprising a residue of isomerized aspartic acid at position 7 [isoD], at a dose of 100 μg, administered in a volume of solution of 200 μl / goal once a month.

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