RU2544957C2 - Method for developing alzheimer disease cell models - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to a method for developing Alzheimer disease cell models for testing medical effectiveness of chemical substances to be further used in medicine, more specifically in treating individual's neurodegenerative diseases caused by neurotoxic effects of beta-amyloid aggregates. A molecular cytotoxic agent is a synthetic analogue of human beta-amyloid 1-42 isomerised in asparagic acid residue in position 7 ([isoD]) (amino acid sequence: DAEFRH[isoD]SGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA). The additional advantage of the present invention is applicability of primary cell cultures as Alzheimer disease models.

EFFECT: advantage of these Alzheimer disease cell models as compared to currently used exogenous-induced models, wherein the cytotoxic effects are caused by a non-modified form of beta-amyloid is a higher ability of the isomerised beta-amyloid to induce both cell necrosis and apoptosis.

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Description

FIELD OF THE INVENTION

The invention relates to the creation of cellular models of Alzheimer's disease, intended for testing the drug efficacy of chemicals with a view to their further use in medicine, more specifically in the treatment of human neurodegenerative diseases caused by the neurotoxic effects of amyloid beta aggregates.

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 (7). 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).

The molecular mechanism of the pathogenic action of aggregated forms of beta-amyloid has not been established, but it is well known that the presence of beta-amyloid aggregates in the tissues of the body causes a pronounced cytotoxic effect (6). Currently, cellular models of Alzheimer's disease are widely used for screening potential drugs (7-8). In this case, both exogenous and endogenous cell models are used, in which the pathology is due to excessive production of human beta-amyloid or tau protein.

US Pat. No. 5,994,084 describes transgenic cell models that, along with the overproduction of tau protein, are characterized by the content of kinases responsible for hyperphosphorylation of tau protein, which causes its increased aggregation. US patent application US 2009/0280110 describes a technique for controlling early cellular phenomena in Alzheimer's disease in which cells containing tau and tubulin proteins are in direct contact with amyloid beta. US Pat. No. 6,803,233 describes an exogenously-induced cell model of Alzheimer's disease in which the primary cell culture from the rodent brain is exposed to various agents, including amyloid beta, to induce in them phenomena associated with AD, such as the formation of neurofibrillary tangles, hyperphosphorylation and / or fragmentation of the tau protein and the production of cytokines TGF-beta, IL-1b or TNF. Patent Application WO 2010/112737 describes a human neural model derived from pluripatent stem cells containing the genome of patients diagnosed with Alzheimer's disease. US 2005/0172344 describes a cell model derived from hippocampal cells isolated from an animal model of AD containing mutations in the gene encoding APP and PS1. US Pat. No. 6,548,261 describes a methodology for testing drugs that are capable of inhibiting cellular phenomena associated with AD based on transfected mammalian neuronal cell lines. At the same time, no information was found on the creation and use of exogenously-induced cell models of Alzheimer's disease using modified forms of amyloid beta.

Previously, the authors of the present invention showed that isomerization of the aspartic acid residue at position 7 leads to zinc-dependent oligomerization of the metal-binding domain of 1-16 beta-amyloid (9). Since amyloid plaques contain up to 75% of isomerized beta-amyloid and an excess of zinc ions, we suggested that it is the zinc complexes of this isoform of beta-amyloid that can be a molecular agent that causes oligomerization and subsequent aggregation of soluble forms of beta-amyloid, i.e. the processes that are considered by most researchers as triggers of the pathogenesis of Alzheimer's disease.

SUMMARY OF THE INVENTION

The present invention relates to a method for creating cellular models of Alzheimer's disease in which a cytotoxic effect is induced by introducing into the culture medium a synthetic analogue of aspartic acid isomerized at the 7 position of human amyloid beta 1-42. The advantage of such cellular models of Alzheimer's disease in comparison with the currently existing exogenously induced models in which the cytotoxic effects are caused by the unmodified form of amyloid beta is the higher ability of isomerized amyloid beta to induce cell death both by necrotic and apoptotic mechanisms. An additional advantage of this invention is the possibility of using primary cell cultures as models of Alzheimer's disease.

Technical result

The technical result achieved using the patented invention is to create exogenously-induced cell models of Alzheimer's disease.

An example embodiment of the invention

An immortalized culture of human neural stem cells NSC-hTERT was used as a cell line (10). NSC-hTERT cells were obtained by immortalization by introducing the telomerase catalytic subunit gene (hTERT) gene into primary culture cells. Primary cultures of human neural stem cells (NSC) were provided by I.N. Saburina (Institute of Gene Biology RAS). Cells were obtained from the anterior brain region of a human embryo (medical abortus) at the age of 9 weeks. The brain of the embryo was mechanically ground, treated with trypsin and Versen solutions, pipetted and transferred to the culture medium. Cells were cultured at 3% oxygen in a growth medium consisting of the following components: DMEM / F12 medium, 2% FetalClone III (Hyclone, USA), 10 ng / ml FGF2, 10 ng / ml EGF, 0.11 mg / ml sodium pyruvate , 0.32 mg / ml glutamine, a single concentration of N2supplement (Invitrogen, USA) and 40 units / ml gentamicin. When cells are maintained in such an environment, they grow like a normal plastic-adhesive culture. Reseeding was carried out upon reaching a monolayer using Versen and trypsin-Versen solutions. With a decrease in the concentration of FetalClone III, cells are able to proceed to growth in the form of neurospheres. Primary cultures of NSC have limited proliferative potential and in our conditions were able to reach a level of about 42 doublings of the population for about six months. Cell transfection was carried out by a lentiviral construct containing hTERT cDNA under the CMV promoter. The design of pLA-CMV-hTERT (9645 base pairs) was kindly provided by P.M. Chumakov (IMB RAS). The viral concentrate (with a titer of 108 / ml) was diluted twice with full growth medium, 5 μg / ml of polybrene was added and added to the growing NSC. Cloning was not performed. The efficacy of lentiviral transfection (by parallel administration of GFP) was about 75%. The age of the cells at the time of transfection was about 9 doublings of the population. In a continuous experiment lasting 560 days, it was shown that NSC-hTERT did not change growth rates and proliferated over 100 doublings of the population. These cells possess telomerase activity, express hTERT (determined using antibodies) and contain somewhat elongated telomeres. They retain a diploid karyotype. They are able to form neurospheres. They express genes associated with the stem state (nestin, determined by RNA and protein), neuronal differentiation (beta-III-tubulin, determined by RNA and protein), neuron-specific enolase, tyrosine hydroxylase, choline acetyl transferase (determined by RNA) and glial differentiation (glial fibrillar acidic protein, determined by RNA and protein) and lipophilin (determined by RNA).

Synthetic peptides Aβ: [H2N] -DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA- [COOH] and isoAβ: [H2N] -DAEFRH [isoD] SGYEVHHQKLVFFAEDVGSNKGAIIGLHVG All experiments and preparations were carried out under sterile conditions. Synthetic peptides were dissolved in dimethyl sulfoxide to a final stock concentration of 10 mM. Then, 1 μl of the effluent was dissolved in 5 μl of deionized water. The resulting solution was added to 500 μl of medium to a final concentration of the peptide of 20 μM. Differentiated nerve cells were used in the experiment; no serum was added to the medium. Cells were cultured in 500 μl of medium in 12-well plates. In the cells, the medium in which the cells were cultured was replaced with a medium containing 1 μl of dimethyl sulfoxide and 5 μl of deionized water (three control cells), Αβ peptide (three cells) and isoAβ peptide (three cells). Then the tablet was placed in a thermostat at 37 ° C for 24 hours.

The following protocol was used to remove cells: growth medium was selected; cells were washed once with Versen's solution (~ 120 μl per 1.9 cm ² ); filled with a monolayer solution for cell removal (0.25% sterile Trypsin solution with Hanks salts (without Ca and Mg)) (~ 120 μl per 1.9 cm ² ); incubated at 37 ° C, periodically gently shaking, ~ 1-2 min; when the cells completely separated from the substrate, 500 μl of complete medium was added and the cells were thoroughly dispersed; counted the number of cells in the counting chamber of Goryaev under an inverted microscope with preliminary trypan blue staining.

Cell cytometric analysis was performed on a GALLIOS flow cytometer (BeckmanCoulter).

Determination of the percentage of cells with a damaged membrane. Cells with a damaged membrane were determined using propidium iodide (PI) (Sigma, USA) (Ex / Em 535/617 nm). For this, propidium iodide was added to the cell suspension 1 minute before the start of measurements to a final concentration of 10 μg / ml. The percentage of cells having fluorescence in the red region of the spectrum was recorded upon excitation by a laser with a wavelength of 488 nm.

Determination of the percentage of apoptotic cells. The percentage of apoptotic cells was determined using Annexin V, which allows the detection of phosphatidylserine output to the cell surface. To determine the percentage of apoptotic cells, the cell suspension was centrifuged (800 rpm, 7 min), the supernatant was taken and resuspended in Annexin V binding buffer (10 mM HEPES, 140 mM NaCl and 2.5 mM CaCl2, pH 7.4). Then, 5 μl of annexin V solution labeled with FITC fluorescein (Invitrogen) with excitation and Ex / Em emission wavelengths of 494 and 518 nm, respectively, was added to 100 μl of a cell suspension of 106 cells / ml. Thus, fluorescence of annexin-positive cells was recorded in the green region of the spectrum when excited by a laser with a wavelength of 488 nm. Incubation was carried out for 15 minutes in the dark at room temperature, after which 300 μl of cold annexin binding buffer was added and put on ice. Viable cells were not stained with annexin or propidium iodide. Annexin stained and unpainted PI cells were characterized as early apoptotic. Stained with PI and Annexin cells were defined as late apoptotic or necrotic. Cells stained with propidium iodide alone were characterized as necrotic.

Determination of the percentage of dying cells. Dying cells include cells with altered morphology, characterized by a decrease in cell size and an increase in their granularity. These parameters during flow cytometry are estimated using the parameters of small angle and lateral scattering. Thus, the percentage of dying cells in the population was determined by estimating the percentage of cells with small size and high granularity.

Determination of the percentage of cells with reduced mitochondrial potential

The percentage of cells with reduced mitochondrial potential was determined using a DilC1 dye (5) (Invitrogen), whose excitation and Ex / Em emission wavelengths were 638 and 658 nm, respectively. Thus, fluorescence of stained DilC1 (5) cells was recorded in the red region of the spectrum upon excitation by a laser with a wavelength of 638 nm. For staining, cells were resuspended in 100 μl of medium to a concentration of 1 × 106 cells / ml and 0.5 μl of 10 μM DilC1 (5) was added to them to a final concentration of 50 nM, after which they were incubated for 20 minutes in a CO ₂ incubator at 37 ° FROM. The dye penetrates the cytosol of cells and accumulates in the mitochondria. Propidium iodide was added 1 minute prior to measurement as described above. The percentage of cells with reduced mitochondrial potential was recorded as the percentage of cells with an intact membrane having a reduced mitochondrial dye fluorescence intensity. The spectral characteristics of the dyes used: Annexin-FITC, DilC1 (5), propidium iodide allow using them for simultaneous staining and analysis of cells on a flow cytometer. First, DilC1 staining is performed (5), and then staining with FITC-labeled Annexin. PI is added to the cells 1 minute before the measurement, as described above.

A cytofluometric study of apoptotic changes and morphological characteristics of cells under the influence of beta-amyloid and its isoform has revealed that the isoform has a greater cytotoxic effect in relation to neuronal cells than beta amyloid. The effect is manifested in the cultivation of cells with intact beta-amyloid (Ab) and its isomerized aspartic acid residue at position 7 of the isoform (isoAb) for 24 hours (Figures 1, 2). Figure 1 shows the percentage of dying cells, estimated as the percentage of cells with small size and high granularity. From the presented data it is seen that the proportion of dying cells under the action of isoform of beta-amyloid increases and is more than 65%.

Figure 2 shows the percentage of early apoptotic cells, that is, cells in the early apoptosis stage, after 24 hours of incubation with amyloid beta and its isoform. As follows from the above data, a significant increase in the percentage of early apoptotic cells to 55% occurs under the action of the amyloid beta isoform.

Thus, human amyloid beta 1-42 (isoAb) isomerized from the aspartic acid residue at position 7 is much more effective than unmodified beta 1-42 amyloid (Ab) and induces both necrosis and apoptosneural cells. Since cell death under the influence of various forms of beta-amyloid plays a key role in Alzheimer's disease, it is clear that isoAb is a more potent agent than Ab than Ab, and cell models of Alzheimer's disease based on the use of isoAbB as an exogenous factor in the induction of cell death seem to be more adequate.

Claims (1)

A method of creating cellular models of Alzheimer's disease, characterized in that the cytotoxic effect is caused by adding to the culture medium a synthetic analogue of the human isomerized aspartic acid residue at position 7 ([isoD]) of human beta-amyloid 1-42 (amino acid sequence: DAEFRH [isoD] SGYEVHHQKLVFFAEDVGGNKGVKGVKNKGVKNVGSNKGVKNVGGNKGVKNVGSNK .

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