KR102203860B1 - Induced pluripotent stem cells having presenilin protein mutants(E120K) derived from Alzheimer's disease patients - Google Patents

Abstract

The present invention relates to an Alzheimer-related disease screening, personalized treatment, and manufacturing method using induced pluripotent stem cells containing a presenilin 1 (PS1) mutant gene having an E120K substitution derived from blood cells.

Description

Induced pluripotent stem cells having presenilin protein mutants (E120K) derived from Alzheimer's disease patients}

Using blood cells that are easier to collect than skin cells, an induced pluripotent stem cell dedifferentiated from human PBMC containing an APP mutant gene having an E120K substitution, which is a presenilin 1 protein mutation, is provided.

Alzheimer's disease is the most common degenerative brain disease that causes dementia, and was first reported by German psychiatrist Dr. Alois Alzheimer in 1907. Alzheimer's disease is characterized by a very slow onset and gradual progression. In the beginning, they mainly show problems with the memory of recent events, but as they progress, they are accompanied by other abnormalities in other cognitive functions such as language and judgment, and eventually all functions of daily life are lost.

Genetic factors have been reported to account for less than 10% of all Alzheimer's incidences, and people with immediate family members who have it have a higher risk of developing it than those who do not.

Alzheimer's disease is a family member of Alzheimer's disease if there are mutations in the amyloid precursor protein gene (located on chromosome 21), presenillin 1 gene (located on chromosome 14), and presenillin 2 gene (located on chromosome 1). Although known, they are all only involved in the onset of premature (early) Alzheimer's disease, which occurs in the 40s to 50s, and is not related to the onset of most late-stage (old) Alzheimer's.

In the case of Alzheimer's disease, existing animal models have an inherent limitation in that it is difficult to reproduce the pathophysiological symptoms directly related to Alzheimer's disease, so the development of treatments or treatments using existing animal models is limited. In other words, in the case of the mouse model, most of the cases of Alzheimer's disease do not appear in humans even though they have mutations in the same gene as those of Alzheimer's disease patients.

After inducing dedifferentiation of skin cells of Alzheimer's disease patients to make induced pluripotent stem cells (iPSCs), they were differentiated into neurons again to investigate their characteristics. As a result, the pathological markers of Alzheimer's disease of each patient can be measured. There have been reports of studies that show statistically significant differences when compared to normal subjects (Inoue et al., 2013; Israel et al., 2012). This iPSC-based research model is expected to be used as a good research model for verifying the efficacy of therapeutic agents that inhibit pathological biomarkers and searching for customized therapeutics tailored to each patient.

Therefore, research and development of induced pluripotent stem cells having characteristics that can be used for screening of new drugs, high biosimilarity, and pathological characteristics of Alzheimer's disease are required.

One aspect relates to induced pluripotent stem cells dedifferentiated from human PBMCs containing a presenilin 1 protein mutant gene having an E120K substitution.

Another aspect relates to a nerve cell obtained by differentiating the stem cell into a nerve cell.

Another aspect is a presenilin 1 protein mutant gene having an E120K substitution comprising the step of introducing a vector expressing a dedifferentiation factor into PDMC containing a presenilin 1 protein mutation having an E120K substitution isolated from the blood of Alzheimer's patients. It relates to a method for producing a stem cell line containing.

Another aspect is a method of screening for a drug effective for preventing or treating Alzheimer's disease, comprising the steps of: contacting the nerve cells with a test substance;

Measuring at least one of Aβ expression amount, phosphorylated tau protein expression amount, imbalance of mitochondrial division and fusion, and increased autophagy of the contacted nerve cells;

Comparing the measured results with the measured results from the nerve cells not in contact with the test substance; It relates to a drug screening method comprising a.

In order to achieve the above object, one aspect relates to an induced pluripotent stem cell dedifferentiated from human PBMCs containing a presenilin 1 protein mutant gene having an E120K substitution.

In the present specification, the term “inducible pluripotent stem cell” refers to a pluripotent stem cell artificially created by artificially expressing a specific gene to induce an already differentiated adult somatic cell. Also called artificial pluripotent stem cells, pluripotent stem cells, pluripotent stem cells, or dedifferentiated stem cells. The induced pluripotent stem cells may be variously differentiated into organ cells such as brain, heart, joint, bone, blood vessel, stomach, liver, skin, kidney, cartilage, intestine, nerve, or pancreas.

In the present specification, the term “PBMC (peripheral blood-derived mononuclear cell)” refers to mononuclear cells derived from peripheral blood isolated from and obtained from Alzheimer's patients. Since they were isolated directly from patients, the genetic and pathological characteristics of individual patients It refers to a peripheral-derived mononuclear cell with characteristics that can reflect all factors. Therefore, a selected therapeutic agent that exhibits an effect on patient-derived pluripotent stem cells can be expected to exhibit excellent effects on that patient as well. The patient-derived induced pluripotent stem cell may be a cell derived from a patient having each disease subtype, and when using the same, it is possible to effectively select a stem cell therapeutic agent having a specific effect on a specific disease subtype.

In the present specification, the “presenilin 1 (PS1) mutant gene” is a gene involved in the development of Alzheimer's disease, and a mutation appears in the presenilin 1 gene on chromosome 14 of humans. The mutant gene may be any mutant gene in which one or more bases are substituted with a normal gene, and may be a mutant gene in which two or more bases are successively or discontinuously substituted, deleted or added, and presenyl Including that the 120th amino acid from the end of the amino group of Lin 1 is substituted with Lysine in Glutamic acid.

In the present specification, Alzheimer's disease caused by the presenilin 1 mutant gene is characterized by accompanying cerebellar ataxia. The cerebellar ataxia is a symptom that occurs due to the causes of cerebellar tumors, vascular disorders, cerebellar constriction, toxicosis, and infectious disease. Standing walking disorder, limb muscle abnormality, muscle tone reduction, inability to cooperative movement, measurement movement disorder, transmutation movement disorder and Holms -It is characterized by causing a stwart phenomenon.

In the present specification, "vector" or "expression vector" refers to a plasmid, viral vector, or other medium known in the art capable of inserting a nucleic acid encoding a structural gene and expressing the nucleic acid in a host cell. Means. Preferably, it may be a viral vector. Examples of the viral vector include, but are not limited to, retroviral vectors, adenovirus vectors, Herpes virus vectors, abipox virus vectors, and lentiviral vectors. In particular, a method using Sendai virus is preferred. The Sendai virus vector is constructed so that all of the viral genes have been removed or altered so that a non-viral protein is produced in the infected cells by the viral vector. The main advantages of Sendai virus vectors for gene therapy are that they deliver large amounts of genes into cloned cells, accurately insert the transferred genes into cellular DNA, and do not cause continuous infection after gene transfection.

The “vector” or “expression vector” can be introduced into cells by a method known in the art. For example, but not limited thereto, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran Transfection using (DEAE Dextran-mediated transfection), transfection using polybrene (polybrene-mediated transfection), electroporation, gene gun, and other known methods for introducing nucleic acids into cells Can be introduced into cells for production of transgenic animals.

The “nerve cell” or “neuron” refers to the structure and functional unit of the nervous system in the human body, and is preferably a neuron or a neuron in the brain.

Another aspect relates to a nerve cell obtained by differentiating the induced pluripotent stem cell into a nerve cell.

Another aspect is a presenilin 1 protein mutant gene having an E120K substitution comprising the step of introducing a vector expressing a dedifferentiation factor into a PBMC containing a presenilin 1 protein mutation having an E120K substitution isolated from the blood of Alzheimer's patients. It relates to a method for producing a stem cell line containing.

Another aspect is a method of screening for a drug effective for preventing or treating Alzheimer's disease, comprising the steps of: contacting the nerve cells with a test substance;

Measuring at least one of Aβ expression amount, phosphorylated tau protein expression amount, imbalance of mitochondrial division and fusion, and increased autophagy of the contacted nerve cells;

It relates to a drug screening method comprising; comparing the measured results with the results measured from the nerve cells not in contact with the test substance.

The time for floating culture of the PBMC may vary depending on the growth rate of cells, and it takes 4 hours in the present invention.

In the process of culturing the induced pluripotent stem cells to differentiate into PS1-E120K iPSC-derived neurons, SB431542 (Reagent Direct), 10uM LDN 193189 (Reagent Direct), brain derived neurotrophic factor (BDNF) and glial cells Various factors such as glial cell-derived neurotrophic factor (GDNF) can be added, and SB431542 (Reagent direct) and LDN 193189 (Reagent direct) are added in the culture process to differentiate EB into neurons. The culture process to induce the can be made for 7 to 8 days.

The term “treatment” refers to, or includes alleviation, inhibition of progression, or prevention of a disease, disorder or condition, or one or more symptoms thereof.

The terms “administering,” “applying”, “introducing” and “implanting” are used interchangeably and in a method or route that results in at least partial localization of a patch or composition to a desired site according to one embodiment. It may mean the placement of a patch or composition according to an embodiment into an individual by.

In the step of administering an Alzheimer's prophylactic and therapeutic candidate substance to the Alzheimer's model cell line, the prophylactic and therapeutic candidate substance is, for example, a test compound or a test composition, a small molecule compound, an antibody, or an antisense nucleotide. (antisense nucleotide), short interfering RNA (siRNA), short hairpin RNA (shRNA), nucleic acids, proteins, peptides, other extracts or natural products.

Raf-1, FAK1, MEK-1, RAD51, RAD52, GADD45 GAMMA, NF-kB p52, Rb2, Oct3, Klf4 in the markers for verifying whether the peripheral blood-derived mononuclear cells are dedifferentiated into induced pluripotent stem cells. SOX2, SSEA4, NANOG and TRA-1-81.

As a specific example, the number and combination of markers selected to confirm the formation of dedifferentiated stem cells are not specifically limited, and various combinations may be selected and used. By measuring the level of expression of the biomarkers at the mRNA or protein level, it is possible to confirm whether or not dedifferentiated stem cells are formed. Since the nucleic acid sequence and protein sequence of the markers of the present invention are already known, detection of a marker for diagnosis can be usefully detected by using an agent capable of measuring mRNA or protein level based on the known sequence. . The detection of the marker protein is performed by contacting a sample with an antibody that specifically recognizes the protein and measuring its antigen-antibody complex formation. The amount of antigen-antibody complex formation can be quantitatively measured through the size of the signal of the detection label. The detection label may be selected from the group consisting of enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules and radioactive isotopes, but is not limited thereto. Known assay methods for measuring protein levels include Western blot, ELISA, radioimmunoassay, radioimmunodiffusion method, octeroni immune diffusion method, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, Competitive or non-competitive analysis methods such as precipitin reaction, gel diffusion presipitin reaction, aggregation assay, fluorescence immunoassay, protein A immunoassay, FACS, protein chip, etc. may be used, but are not limited thereto. Can be done by In the above, ELISA is a direct sandwich ELISA using another labeled antibody that recognizes an antigen in a complex of an antibody and an antigen attached to a solid support, and reacts with another antibody that recognizes an antigen in a complex of an antibody and an antigen attached to a solid support. It includes various ELISA methods such as indirect sandwich ELISA using a labeled secondary antibody that recognizes this antibody.

By using blood cells that are easier to collect than skin cells, and using induced pluripotent stem cells of Alzheimer's patients with a mutation in the presenilin 1 protein, we provide a method of screening customized treatments suitable for individual disease characteristics and treatments thereof. .

1 is a diagram showing the expression results of undifferentiated markers such as OCT4, SOX2, Nanog, SSEA4, and Tra-1-81 of iPSCs established from PS1-E120K mutant patients.
2 is a diagram showing the genomic DNA sequencing results of PS1-E120K iPSC.
Figure 3 shows the immunocytochemical analysis results showing the differentiation ability of iPSCs forming three embryonic layers (triderm) including ectoderm (TUJ1, green), mesoderm (SMA, green), and endoderm (AFP, red) It is a drawing.
4 is a diagram showing the results of chromosomal karyotyping analysis of PS1-E120K iPSC and a control group.
5 is a diagram showing the results of reverse transcription PCR analysis showing that there is no Sendai virus vector or mycoplasma remaining in the cell.
6 is a diagram showing the expression results of neuronal progenitor cell markers of SOX2/DAPI, Nestin/DAPI and Musashi/DAPI of neuronal progenitor cells differentiated from PS1-E120K iPSC.
7 is a diagram showing the expression results of neuronal markers such as TUJ1/DAPI, MAP2/DAPI, TBR1/CTIP2 and ChAT/MAP2 in cortical neurons differentiated from PS1-E120K iPSC.
8 is a view showing the result that there is no significant difference in the neural cell differentiation tendency between the control and PS1-E120K iPSC.
9 is a diagram showing the results of detection of the expression levels of extracellular Aβ ₄₂ and Aβ ₄₀ cultured in PS1-E120K iPSC-derived neurons by ELISA.
Figure 10 shows the difference in the amount of APP expression in the control and PS1-E120K iPSC-derived neurons. 6E10 (green), 4G8 (red), and TUJ1 (with an antibody against Aβ ₄₂ (red) at 10 weeks of neuronal differentiation) Green) and DAPI (blue) is a diagram showing the results of immunocytochemical analysis showing the expression of anti-stained Aβ precipitate.
11 is a view showing the difference in the amount of APP expression in the control and PS1-E120K iPSC-derived neurons through Western blot and ELISA analysis.
12 is a view showing the difference between the expression levels of p-tau, AT8 and AT180 in the control and PS1-E120K iPSC-derived neurons through Western blot and ELISA analysis.
13 is a view showing the results of immunocytochemical analysis antistained with AT8 (red), MAP2 (green), and DAPI (blue) in control and PS1-E120K iPSC-derived neurons at 10 weeks of neuronal differentiation.
14 is a diagram showing the difference in the expression levels of MAP2, AT8 (neuronal cell body) and AT8 (neurite) in the control and PS1-E120K iPSC-derived neurons through ELISA analysis.
Figure 15 is a control and PS1-E120K iPSC-derived neurons in mitochondrial fusion (OPA1, Mfn1 and Mfn2) and cleavage (Drp1, p-Drp1 and Fis1) related protein, mitophagy (PINK1 and PARKIN) related protein expression levels It is a diagram showing the difference through Western blot and ELISA analysis.
16 is a diagram showing changes in the expression level of m-RNA associated with mitochondrial fusion and division in control and PS1-E120K iPSC-derived neurons at 10 weeks of neuronal differentiation by Q-PCR.
17 is a view showing differences in the expression levels of Ub, p62, Beclin1, LC3B and LAMP2 proteins in the control and PS1-E120K iPSC-derived neurons through Western blot and ELISA analysis.
18 is a diagram showing the difference in the expression levels of proteins stained with LC3B (red) and DAPI (blue) in the control and PS1-E120K iPSC-derived neurons through immunocytochemical, Western blot and ELISA analysis.
Figure 19 shows the difference in the expression levels of autophagy-related proteins stained with Cyto ID (green) and DAPI (blue) in the control and PS1-E120K iPSC-derived neurons, immunocytochemical, and the number and size of autophagy vesicles through ELISA analysis. It is a figure shown.

Example 1: Manufacturing process of induced pluripotent stem cells

Two blood samples were taken from a 38 year old AD patient and a 72 year old normal elderly patient. Monocytes (MNCs) were freshly isolated from peripheral blood of PS-E120K patients using the Ficoll-Paque™ PLUS method (GE Healthcare, USA). The isolated peripheral-derived mononuclear cells (PBMCs) were suspended in monocyte culture for 4 days, and then dedifferentiated using Sendai virus vector (SeVdp) expressing four dedifferentiation factors (OCT4, SOX2, cMYC, KLF4). Three or more individual clones were picked for iPSC generation and the best growing clone was selected for further analysis. The selected clones are cultured for 4 weeks to form an induced pluripotent stem cell colony. No significant differences in age-related characteristics were observed between the two iPSCs (PS1-E120K iPSCs in 38-year-old AD patients and iPSC controls in 72-year-old normal elderly patients).

Example 2: Induction of differentiation of induced pluripotent stem cells into embryonic bodies

The human iPSC of Example 1 was obtained using 0.5% TrypLE Select (Thermo Fisher) and an embryoid body (EB, Embryoid body) was formed. Separated embryoid bodies were transferred to 96-well Nunclon ^TM Sphera ^TM microplates (Thermo Fisher), the medium was exchanged every other day, and plated on glass coverslips coated with 0.2% gelatin on the 8th day. Then, EB was spontaneously differentiated for an additional 8 days.

Example 3: Differentiation of induced pluripotent stem cells into cortical neurons

For the differentiation of neurons in the cerebral cortex, the first embodiment of the iPSCs were aggregated rapidly from the cells were dissociated into a single 96-well Nunclon ^{TM TM} Sphera microplate (Thermo Fisher). EB was incubated for 8 days for differentiation into the nervous system in DMEM/F12 medium. These nerve-induced EBs were dissociated using Accutase ^™ (Stem Cell Technologies) and seeded on dishes coated with 15 μg/ml poly-L-ornithine (Sigma) and 5 μg/ml laminin (Sigma). Once neural precursor cells (NPCs) are formed, 0.1 M non-essential amino acids (NEAA), 0.1 M 2-mercapto ethanol, 0.1 antibacterial-stimulating solution, 1% D-Glucose (Life Technologies), vitamin A It was cultured in DMEM/F12 medium containing no 1% B27 supplement (all Thermo Fisher), 200 mM L-glutamine (Sigma), and 20 ng/ml basic fibroblast growth factor (bFGF, PeproTech Korea). Lastly, NPC was used as a vitamin A-free 1% B27 supplement, 1x Glutamax (all Thermo Fisher), 10 ng/ml brain derived neurotrophic factor (BDNF), and 10 ng/ml glial-derived nerve. Nutrient factor (glial cell-derived neurotrophic factor, GDNF), 10 ng/ml neurofinlin-3 (NT3) (PeproTech Korea) was differentiated into cortical neurons for 10 weeks in Neurobasal A medium placed in a PLO/laminin coated dish. .

In the following experimental examples, immunocytochemical analysis was performed under the following conditions.

iPSCs were fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature (RT). Fixed cells were washed with TPBS containing 0.1% TritonX-100 (Sigma) in phosphate buffered saline (PBS) to increase permeability, and blocked with 5% normal horse serum (Vector Labs) for 30 minutes at room temperature. . Thereafter, the solution was incubated with the primary antibody while blocking the solution overnight while gently shaking at 4°C. After washing three times with TPBS, incubation with a secondary antibody (Thermo Fisher) for 90 minutes at room temperature, and then counter staining for 15 minutes using DAPI. All fluorescence images were imaged by confocal microscope (TCSSP5II, Leica). The following primary antibodies were used: anti-OCT4 (1:200, Santa Cruz), anti-SOX2 (1:200, Millipore), anti-NANOG (1:200, R&D Systems), anti-SSEA-4 ( 1:100, Developmental Studies Hybridoma Bank)), anti-TRA-1-81 (1:100, CHEMICON), Tuj1 anti-tubulin beta III isoform (1:200, Millipore), anti-SMA (1:100, DAKO ); anti-AFP (1:100, DAKO), anti-Nestin (1:200, R&D Systems), anti-Musashi (1:200, Millipore), anti-Map2 (1:200, Millipore), anti-TBR1 (1 :100, Abcam), anti-CTIP2(1:100, Abcam), 6E10 anti-Amyloid "(1:500, Covance), AT8 anti-p-tau (1:1000, Thermo Fisher), anti-ChAT (1 :200, Millipore) and anti-LC3B (1:500, Cell Signaling).

In the following experimental examples, ELISA was performed under the following conditions. The conditioned medium (CM) was collected from neurons (1×10 ⁵ ) cultured for 48 hours after the last medium exchange after 6 and 10 weeks of differentiation. Pellets of iPSC-derived neurons were resuspended in RIPA buffer (Biosesang) containing a protease inhibitor (Thermo Fisher) and a phosphatase inhibitor (Thermo Fisher). It was then cooled for 30 minutes and sonicated. The supernatant was collected after centrifugation at 13,000 rpm for 30 minutes to remove membrane lipids. Protein concentration was measured in a total of 1 μg protein of neurons differentiated for 10 weeks using the BCA Protein Assay Kit (Thermo Fisher) to measure the expression levels of Aβ ₄₂ and Aβ _{40 in} cells. Aβ ₄₀ and Aβ ₄₂ expression levels were measured using a human Aβ ₄₀ and Aβ ₄₂ ELISA kit according to the manufacturer's instructions (IBL), and Aβ ₄₀ and Aβ ₄₂ expression levels were quantified using an ELISA plate leader (BioTek).

Western blot in the following experimental example was performed under the following conditions. The conditioned medium (CM) was collected from neurons (1×10 ⁵ ) cultured for 48 hours after the last medium exchange after 6 and 10 weeks of differentiation. Pellets of iPSC-derived neurons were resuspended in RIPA buffer (Biosesang) containing a protease inhibitor (Thermo Fisher) and a phosphatase inhibitor (Thermo Fisher). It was then cooled for 30 minutes and sonicated. The supernatant was collected after centrifugation at 13,000 rpm for 30 minutes to remove membrane lipids. Protein concentration was measured using the BCA Protein Assay Kit (Thermo Fisher), and 25 ug of protein for each sample was electrophoresed on an 8% or 12% gel. The separated sample was transferred to a PVDF membrane and incubated with the target antibody. Protein bands were expressed by Immobilon Western (Millipore) and detected using Chemi-Doc equipment (Bio-Rad). The following primary antibodies were used: Tau5 anti-tau (1:1,000, Thermo Fisher), AT8 anti-p-tau (1:1,000, Thermo Fisher), anti-OPA1 (1:1,000, Santa Cruz), anti -Mfn1(1:1,000, Abcam), anti-Mfn2(1:1,000, Cell Signaling), anti-Drp1(1:1,000, Cell Signaling), anti-Fis1(1:1,000, Santa Cruz), anti-Ub( 1:4,000, Santa Cruz), anti-LC3B(1:1,000, Cell Signaling), anti-LAMP2(1:1,000, Santa Cruz), anti-Beclin1(1:1,000, Cell Signaling), p62 anti-SQSTM1(1 :1,000, Santa Cruz), 4G8 anti-APP (1:500, Covance), anti-p-Drp1 (Ser637, 1:1000, Cell Signaling) and anti-beta-actin (1:10000,Santa Cruz)

Experimental Example 1: Whether PS1-E120K mutation was established in iPSC

Immunocytochemical analysis was performed to confirm the establishment of the PS1-E120K mutation in iPSCs. In addition, karyotype and chromosome analysis were performed to confirm the establishment of the PS1-E120K mutation in iPSCs. Karyotype analysis was performed through GTG-banding analysis. The iPSC pellet was incubated at 55° C. for 3 hours in a lysis buffer containing 100mM Tris-HCl (pH 8.0), 50mM EDTA, 0.2% SDS, 200mM NACL, and 200µg/ml Proteinase K. Genomic DNA was separated by isopropyl alcohol precipitation followed by a 70% ethanol wash step. Genotyping of the PS1-E120K single nucleotide mutation was performed by PCR amplification of the isolated iPSC genomic DNA, followed by DNA sequencing (Cosmo Genetech, Korea). Total RNA was manually isolated using TRIzol reagent (Life Technologies) lysis and isopropyl alcohol precipitation. Complementary DNA was synthesized using a cDNA synthesis kit (Cosmo Genetech, Korea). Q-PCR analysis was performed using TB Green ^™ Premix Ex Taq ^™ (TaKaRa) with 100 ng/ul cDNA for each sample.

To confirm the establishment of the PS1-E120K mutation in iPSCs, an analysis was performed on the presence of Sendai virus and mycoplasma.

After total RNA isolation and cDNA synthesis, RT-PCR for Sendai virus detection was performed using a final volume of 20 μl at a concentration of 200 ng/ul for each sample, and nuclear protein (NP) remains of Sendai virus vector (SeVdp) Was done to detect whether or not. In addition, PCR-based e-Myco ^TM VALiD Mycoplasma detection kit (iNtRON, Korea) was used to investigate mycoplasma infection in cell culture. PCR was performed using a final volume of 20 ul containing 100 ng/ul of each sample.

As shown in FIG. 1, undifferentiated markers such as OCT4, SOX2, Nanog, SSEA4, and Tra-1-81 of iPSCs established from PS1-E120K mutant patients were expressed.

As shown in FIG. 2, as a result of DNA sequencing of PS1-E120K iPSC, the genotype of PS1-E120K mutant DNA could be observed.

As shown in Figure 3, the differentiation ability of iPSCs forming three embryonic layers (triderm) including ectoderm (TUJ1, green), mesoderm (SMA, green), and endoderm (AFP, red) can be observed in vitro. Could

As shown in Figure 4, it was possible to observe the difference between the chromosomal karyotype of the PS1-E120K iPSC and the control group.

As shown in FIG. 5, it was observed that there was no Sendai virus vector or mycoplasma remaining in the cells by reverse transcription PCR analysis.

Experimental Example 2: Confirmation of differentiation of PS1-E120K mutant iPSC into the nervous system

This experiment was performed to confirm whether the PS1-E120K mutant iPSC was differentiated into the nervous system. iPSCs were aggregated rapidly from the cells were dissociated into a single 96-well Nunclon ^{TM TM} Sphera microplate (Thermo Fisher). EB (embryoid body) is 20% knockout ^TM serum replacement (KSR), 0.1M non-essential amino acids (NEAA), 0.1M 2-mercapto ethanol, 0.1% antibacterial-promoting solution (all purchased from Thermo Fisher), 10uM It was cultured in DMEM/F12 containing SB431542 (Reagent Direct) and 10uM LDN 193189 (Reagent Direct) for 8 days while inducing differentiation into neurons. These nervous system-inducing EBs were dissociated using Accutase ^™ (Stem Cell Technologies) and were coated on a culture dish coated with 15 μg/ml poly-L-ornithine (Sigma) and 5 μg/ml laminin (Sigma).

Once neural progenitor cells (NPCs) are formed, 0.1M non-essential amino acids (NEAA), 0.1M 2-mercapto ethanol, 0.1% antibacterial-promoting solution, 1% DGlucose (Life Technologies), 1% B27 without vitamin A Supplements (all purchased from Thermo Fisher), 200mM L-Glutamine (Sigma) and 20ng / ml basic fibroblast growth factor (bFGF, PeproTech Korea) containing DMEM / F12 culture medium containing.

Finally, 1% B27 supplement without vitamin A in dishes coated with PLO/laminin, 1x Glutamax (Thermo Fisher), BDNF 10 ng/ml, GDNF 10 ng/ml, Neurotropin-3 (NT3) 10 ng/ml (all (Purchased from PeproTech Korea) in Neurobasal A medium, NPCs were differentiated into cortical neurons for 10 weeks.

As shown in FIG. 6, expression of neuroprecursor markers such as SOX2/DAPI, Nestin/DAPI, and Musashi/DAPI could be observed in the neuronal progenitor cells differentiated from PS1-E120K iPSC.

As shown in FIG. 7, expression of neuronal markers such as TUJ1/DAPI, MAP2/DAPI, TBR1/CTIP2 and ChAT/MAP2 could be observed in cortical neurons differentiated from PS1-E120K iPSC. In addition, a high level of neuronal differentiation was observed in this experimental example, and the following is the relative percentage of each neuron type (control vs. PS1-E120K iPSC-derived neurons). MAP2 (80.24% ± 2.47 vs. 80.45% ± 1.81), TBR1 (61.29% ± 2.23 vs64.69% ± 3.47) and CTIP2 (59.65% ± 3.28 vs 49.40% ± 6.73), of which TBR1 / CTIP double positive The proportion of neurons was relatively high at 58.02% ± 4.43 vs 48.41% ± 6.93, indicating that cortical neuron differentiation was efficient. In addition, high levels of ChAT (74.25% ± 6.69 vs 81.20% ± 2.23), a marker for cholinergic neurons, were observed.

As shown in FIG. 8, there was no significant difference in the neural cell differentiation tendency between the control and PS1-E120K iPSCs.

Experimental Example 3: Confirmation of increase in Aβ and phosphorylated tau protein in PS1-E120K mutant iPSC-derived neurons

In order to analyze the functional aspects of familial AD (PS1-E120K), the amount of Aβ secretion was investigated from iPSC-derived neurons in the conditioned medium 6 and 10 weeks after differentiation. The expression levels of Aβ ₄₀ and Aβ ₄₂ were investigated 48 hours after the last medium exchange.

ELISA analysis, as shown in Figure 9, PS1-E120K and in iPSC-derived neurons compared to the extracellular (extracellular) Aβ ₄₂ and Aβ ₄₀ expression amount of a control culture increased by Aβ ₄₂ expression amount is more than two times dramatically. However, no difference in the expression level of Aβ ₄₀ was detected, and the ratio of Aβ ₄₂ / Aβ ₄₀ was significantly increased more than 2 times.

In the PS1-E120K iPSC-derived neurons, intracellular Aβ ₄₂ and Aβ ₄₀ expression levels were significantly increased compared to the control group, Aβ ₄₂ expression level and Aβ ₄₂ / Aβ ₄₀ ratio.

As shown in the immunocytochemical analysis of FIG. 10, the amount of APP expression was significantly increased in PS1-E120K iPSC-derived neurons compared to the control group.

As shown in the Western blot and ELISA analysis results of FIG. 11, the PS1-E120K iPSC-derived neurons significantly increased the amount of APP expression compared to the control group.

Experimental Example 4: Confirmation of increase in phosphorylated protein of PS1-E120K mutant iPSC-derived neurons

The tau protein is generally present in a dissolved form in axons, but is accumulated in dendrites and cell bodies as they become hyperphosphorylated. To analyze phosphorylated tau levels in iPSC-derived neurons, antibodies against AT180 (Thr231 phosphorylated), p-Tau (Ser400, Thr403/Ser404 phosphorylated) and Tau5 (total tau) were used. Then, Western blot analysis was performed. As shown in FIG. 12, it was found that there are multiple bands representing the fractions of AT8, AT180 and p-tau, and that the expression of each protein is strongly increased in PS1-E120K iPSC-derived neurons compared to the control. These findings are consistent with previous studies that reported that brain tissue from Alzheimer's patients in severe Braak stage and neurons with PSEN1 and APP mutations exhibit multiple bands of p-tau (> 50 kDa) compared to controls. do.

In addition, there was an increase in the total expression level of tau (Tau5), indicating that it was associated with a robust increase in the aggregated tau fraction. Since an increase in fractionated tau in PS1-E120K iPSC-derived neurons was found at week 10 of differentiation, tau ELISA was performed to detect soluble tau using conditioned medium (CM). In order to further investigate p-tau accumulation at the cellular level, immunocytochemical analysis was performed using an antibody against AT8. As shown in Figs. 13 and 14, unlike the control, AT8 expression of PS1-E120K iPSC-derived neurons (soma).

Experimental Example 5: Confirmation of imbalance of mitochondrial fusion and division in PS1-E120K mutant iPSC-derived neurons

To investigate whether the balance between mitochondrial cleavage and fusion was impaired in PS1-E120K iPSC-derived neurons, the mitochondrial cleavage-related protein Drp1 (dyinamine-related protein 1), p-Drp1 (for detection of inhibitory phosphorylation of Drp1 in Ser637) and Fis1 (mitochondrial cleavage 1 protein); And the expression levels of the fusion-related proteins OPA1 (optic nerve atrophy 1 gene protein), Mfn1 (membrane protein mitofusin 1), and Mfn2 (membrane protein mitofusin 2).

As a result of Western blot, as shown in FIG. 15, compared to the control group, the significant increase in Drp1 expression in PS1-E120K iPSC-derived neurons, the decrease in the inhibitory p-Drp1 (Ser637), and the corresponding p-Drp1 (Ser637)/Drp1 A decrease in the ratio and a slight increase in Fis1 could be observed. In addition, it was found that the expression of OPA1, Mfn1 and Mfn2 was significantly reduced in PS1-E120K-derived neurons when compared to the control group.

As a result of Q-PCR analysis, as shown in FIG. 16, the increase in the expression of the division-related gene (DRP1) and the decrease in the expression of the fusion-related gene (Mfn1) in the PS1-E120K iPSC-derived neuron was dramatically compared with that of the control neuron.

In addition, mitophagy-related proteins PINK1 and PARKIN were dramatically increased in PS1-E120K-derived neurons compared to the control group. These results strongly suggest that PSEN1 mutations can lead to impaired mitochondrial division and fusion.

Experimental Example 6: Confirmation of inhibition of mitochondrial autophagy function in PS1-E120K mutant iPSC-derived neurons

Defects in autophagy function are known to occur in AD, which is associated with the presence of neurocapillaries and fibrotic tau. It was previously shown that PS1 is required for the degradation of abnormal proteins using the autophagy lysosome system. To further study this in PS1-E120K-derived neurons, the expression levels of autophagy-related proteins were measured as follows: ubiquitin (Ub), p62 (SQSTM1, siquestosome 1; transport protein marker), Beclin1 (autophagy). Phagosome membrane formation related protein), LC3b (light chain 3; autophagosome formation marker) and LAMP2 (lysosomal membrane related protein).

As a result of Western blot analysis, as shown in FIG. 17, the expression levels of Ub and LC3b-II (active LC3b) were significantly increased in PS1-E120K iPSC-derived neurons. As a result of performing immunocytochemical analysis to investigate LC3B expression at the cellular level, it was confirmed that the PS1-E120K iPSC-derived neurons showed a dramatic increase in LC3b around the soma region compared to the control as shown in FIG. did. In contrast, the chaperone-mediated autophagy and lysosome membrane marker, LAMP2, showed a dramatic decrease in PS1-E120K iPSC-derived neurons.

To further investigate the impaired function of autophagy, an autophagy flux assay was performed with the treatment of 10uM chloroquine (CQ), a compound known to interfere with lysosome function. As shown in Figure 18, after CQ treatment in the PS1-E120K iPSC-derived NPC, LC3B-II expression was significantly increased compared to the control group.

In addition, as a result of performing staining in a live cell with Cyto ID, as shown in FIG. 19, the NPC extracted from PS1-E120K iPSC dramatically increased the number of autophagy compared to the control cells. This shows that the PS1-E120K mutation causes autophagy and changes in lysosome expression in differentiated neurons.

Claims (14)

delete delete delete delete delete delete delete delete delete delete delete delete As a method for screening a drug effective for preventing or treating Alzheimer's disease accompanying cerebellar ataxia,
Contacting neurons obtained by differentiating induced pluripotent stem cells dedifferentiated from human PBMCs containing a presenilin 1 (PS1) mutant gene having an E120K substitution isolated from the blood of Alzheimer's patients with a test substance;
Measuring at least one of high expression levels of beta amyloid, high expression levels of phosphorylated tau, mitochondrial dyskinesia, imbalance of mitochondrial division and fusion, and increased autophagy of the contacted neurons;
Comprising the step of comparing the measured results with the results measured from the nerve cells not in contact with the test substance. The method according to claim 13, wherein when one or more of the high expression levels of beta amyloid, high expression levels of phosphorylated tau, mitochondrial dyskinesia, imbalance of mitochondrial division and fusion, and increased autophagy appear, the test substance is reduced. The method further comprising the step of selecting as an effective drug for preventing or treating Alzheimer's disease accompanying cerebral ataxia.

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