JP7305156B2 - Maturation Accelerator of Cells - Google Patents

Description

TECHNICAL FIELD The present invention relates to a screening method for cell maturation-promoting agents and therapeutic agents for neurodegenerative diseases.

Late-onset neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson's disease, and Alzheimer's disease are diseases in which specific neuronal groups in the central nervous system die, and the causes are still unknown. not Model studies are needed to develop therapeutic agents for these neurodegenerative diseases.

In addition, fibroblasts can be mentioned as cultured cells for research with high versatility. In order to study the aging mechanism in vivo using such cells, it is important to develop a technique for rapidly inducing cell aging.

In neurodegenerative disease model studies using nervous system cells induced to differentiate from patient iPS cells, a relatively long culture period is required for maturation of the induced nervous system cells, and long-term culture of 70 to 100 days is required. Even if they do, it is often difficult to observe disease-specific phenotypes (abnormal protein aggregation, etc.) that take decades to develop in humans. In order to solve this problem, methods such as external gene introduction (progerin gene; progerin) (Non-Patent Document 1) and inhibition of telomere synthase (Non-Patent Document 2) are used to improve the aging of iPS cell-derived neurons. techniques have been reported to promote

Cell Stem Cell. 2013 Cell Stem Reports. 2016

However, these methods require specific techniques for gene introduction, are not simple, and their effects are not so strong, so they can only weakly promote cell senescence.
Accordingly, an object of the present invention is to provide a drug that promotes the maturation and senescence of various cells, and a screening method for a therapeutic drug for neurodegenerative diseases using the drug.

Therefore, the inventors of the present invention used a culture system of nerve cells or fibroblasts to search for low-molecular-weight compounds that promote maturational senescence of cells. Found it. Furthermore, if the iPS cell-derived neurons of a Parkinson's disease patient are cultured in a medium containing KU-60019, the Parkinson's disease phenotype in the neurons can be detected after 7 days of culture. They found that it is useful as a drug screening model, and completed the present invention.

That is, the present invention provides the following inventions [1] to [8].

[1] A cell maturation senescence accelerator containing KU-60019 as an active ingredient.
[2] The aging-promoting agent of [1], wherein the cells are nerve cells or fibroblasts.
[3] The mature aging accelerator of [1] or [2], wherein the cells are derived from pluripotent stem cells.
[4] A method for promoting maturation and senescence of cells, which comprises culturing cells in a medium containing KU-60019.
[5] The method of accelerating mature aging according to [4], wherein the cells are nerve cells or fibroblasts.
[6] The method of promoting mature senescence of [4] or [5], wherein the cells are derived from pluripotent stem cells.
[7] A screening method for a therapeutic agent for neurodegenerative diseases, which comprises culturing nerve cells in a medium containing a test substance and KU-60019.
[8] The screening method of [7], wherein the neurons are derived from pluripotent stem cells.

When the agent for promoting cell maturation and senescence of the present invention is used, nerve cells and fibroblasts differentiation-induced from pluripotent stem cells typified by iPS cells undergo maturation and senescence in an extremely short period of time. In addition, if iPS cells derived from neurodegenerative disease patients are cultured in a medium containing the mature aging-accelerating agent of the present invention, the neurodegenerative disease phenotype can be detected in an extremely short period of time. Screening of therapeutic agents for neurodegenerative diseases becomes possible. By acting on fibroblasts to age them, senescent cells can be obtained in a shorter period of time than in the past, making it possible to efficiently conduct aging research.

Fluorescence intensity of a fluorescent reporter (GFP-Synapsin) in mature neurons is shown. DMSO indicates dimethylsulfoxide, KU60019 indicates KU-60019, and ACM indicates astrocyte conditioned medium. Fluorescence intensity of a fluorescent reporter (GFP-Synapsin) in mature neurons is shown. DMSO, KU-60019 are the same as in FIG. The culture conditions and schedule of Example 2 are shown. FGF2: 20 ng/μL, SB431542 (SB): 2 μM, Purmorphamine (PM): 2 μM, CHIR99021 (CHIR): 3 μM. DAPT: 10 μM, GDNF: 20 ng/μL, TGFβ: 1 ng/μL, ascorbic acid (AA): 0.2 mM, dbcAMP (cAMP): 0.5 mM. Co-immunostaining results using bIII tubulin (green) and Nestin (red) antibodies are shown. Quantification results of Nestin-positive cell area are shown. Quantification results of tyrosine hydroxylase (TH)-positive cells are shown. Fig. 2 shows changes in NAD ⁺ concentration in iPS cell-derived neurons when KU-60019 was added. FIG. 10 shows the results of confirming the aging phenotype of iPS cell-derived dopamine neurons (DAN) when KU-60019 was added. FIG. (A to C) Effect of DAN on nuclear membrane structure with and without KU-60019 addition. LaminB1 (green), TH (red). (DE) Observation of DNA damage in DAN with and without KU-60019. γH2AX (green), TH (red). The results of application to Parkinson's disease-iPSCs are shown (measurement of the number of TH-positive cells in PARK4-iPSCs-derived DAN culture). KU60019 addition shows generation of senescence-specific nuclear membrane abnormalities in human fibroblasts. KU60019 addition shows an increase in senescence-specific marker SA-Gal in human fibroblasts. KU60019 addition shows promotion of senescent cells in SHSY5 cells. Fig. 2 shows the neuronal maturation promoting effect of KU60019, KU55933 and NU7441 (same method as in Example 1).

The active ingredient of the cell maturation promoting agent of the present invention is KU-60019.

KU-60019 is a compound represented by the following formula (1).

KU-60019 is known to be an ATM inhibitor, and ATM is the causative gene for the progeria, ataxia telangiectasia. In recent years, it has been reported that in ATM-deficient mice and ATM-deficient nematodes, a decrease in intracellular NAD ⁺ induces neuronal cell death and shortens lifespan (Cell Metabolism. 2016). However, the effect of KU-60019 on promoting the maturation and senescence of nerve cells occurs after about 7 days of culture, so it cannot be explained only by the ATM inhibitory action.
In addition, the excellent effect of promoting mature senescence as in the present invention is specific to KU-60019, since it cannot be obtained with other ATM inhibitors KU55933 or DNA-PK inhibitors NU7441.

Cells that promote mature senescence in the present invention may be cells that can be cultured, such as nerve cells, fibroblasts, Hela cells (cells derived from human cervical cancer), and SHSY5 (human neuroblastoma) cells. is preferred.
The nerve cells of the present invention may be of any type, but are preferably nerve cells involved in neurodegenerative diseases. For example, using dopaminergic neurons, it is possible to screen for Parkinson's disease, a neurodegenerative disease that reduces cognitive function.
In addition, the fibroblasts of the present invention may be normal fibroblasts, and skin-derived fibroblasts are preferable due to their easy availability.

In the present invention, the cells targeted for promoting mature senescence are preferably cells derived from pluripotent stem cells. Pluripotent stem cells include embryonic stem cells (ES cells), ntES cells and iPS cells, with iPS cells being more preferred. Among iPS cells, human iPS cells are preferred. iPS cells derived from patients with cytopathic disease are preferable as iPS cells used for screening therapeutic agents for cytopathic disease.

A neurodegenerative disease in the present invention is a disease in which specific neuronal cell groups in the central nervous system are gradually damaged and lost. Examples include amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, progressive supranuclear palsy (PSP), Hanchileton's disease, multiple system atrophy, spinocerebellar degeneration (SCD) and the like.

In the present invention, cells are cultured in a medium containing KU-60019 to promote maturational senescence of cells.

The concentration of KU-60019 in the medium is preferably 0.1 μM to 50 μM, more preferably 0.5 μM to 10 μM, from the viewpoint of promoting mature senescence.

In addition, although the medium used for culture differs depending on the differentiation process of the cells used, KBM Neural Stem Cell (KOHJIN BIO) is used as the basal medium. Fibroblast growth factor (FGF2), TGF-β type I receptor inhibitors (SB431542, etc.), ROCK inhibitors (Y27632, etc.), Sonic Hedge Hog pathway agonists (Purmorphamine, etc.) are added to the basal medium from stem cells to nerve cell generation. , a GSKβ inhibitor (CHIR99021, etc.) or the like is preferably added for culturing.
The concentration of fibroblast growth factor in the medium is preferably 10-40 ng/μL, more preferably 20 ng/μL. The concentration of SB431542 in the medium is preferably 1-5 μM, more preferably 2 μM. The concentration of Y27632 in the medium is preferably 1-50 μM, more preferably 10 μM. The concentration of Purmorphamine in the medium is preferably 1-5 μM, more preferably 2 μM. The concentration of CHIR99021 in the medium is preferably 1-8 μM, more preferably 3 μM.
Culture conditions from stem cells to nerve cells are more preferably 5% CO ₂ , 4% O ₂ and 35-40°C.

In addition, to induce differentiation from neurons to neurons and to mature senescence, the basal medium may contain a γ-secretase inhibitor (e.g., DAPT), glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), It is preferable to culture with the addition of transforming growth factor (TGFβ), dbcAMP, ascorbic acid (AA), or the like.
Here, the concentration of DAPT in the medium is preferably 1-50 μM, more preferably 10 μM. The concentration of GDNF in the medium is preferably 10-50 ng/μL, more preferably 20 ng/μL. The concentration of BDNF in the medium is preferably 10-50 ng/μL, more preferably 20 ng/μL. The concentration of TGFβ in the medium is preferably 0.2-5 ng/μL, more preferably 1 ng/μL. The concentration of dbcAMP in the medium is preferably 0.1-3 mM, more preferably 0.5 mM. The concentration of AA in the medium is preferably 0.1-1 mM, more preferably 0.2 mM.
More preferably, culture conditions for accelerated maturation and senescence of nerve cells are 5% CO ₂ , 4% O ₂ and 35-40°C. More preferably, the culture conditions for accelerating maturation and senescence of fibroblasts are 5% CO ₂ and 35 to 40°C.

When using a medium supplemented with KU-60019, 7 to 17 days are sufficient for the differentiation of stem cells, eg, neuronal cell aggregates (neurospheres) into neurons. In addition, when a medium supplemented with KU-60019 is used, 7 to 17 days is sufficient for the differentiation from neuronal cells to mature senescence of neuronal cells. The differentiation from stem cells to nerve cells can be confirmed by reducing Nestin and quantifying tyrosine hydroxylase-positive cells, which are specific markers of dopaminergic neurons (DAN). In addition, maturational senescence of nerve cells can be confirmed by, for example, disruption of LaminB and excessive DNA damage.

The method of screening for therapeutic agents for neurodegenerative diseases of the present invention can be carried out by culturing nerve cells in a medium containing a test substance and KU-60019. The medium and culture conditions are the same as in the method for accelerating maturation and senescence of nerve cells. A predetermined amount of the test substance may be added and cultured.
By comparing the degree of mature aging of nerve cells when the test substance is added with that when the test substance is not added, the effect of the test substance on suppressing nerve cell aging can be evaluated.

The present invention will now be described in more detail with reference to examples.

Example 1
A commercially available inhibitor library was used to screen low-molecular-weight compounds that promote the differentiation and maturation of neurons according to the following procedure, and the compound KU-60019 that promotes the maturation of neurons induced from human iPS cells was identified.
(1) Neural cell clusters (Neurospheres) containing neural stem cells were prepared from iPS cells by suspension culture, passaged 6 times, and differentiated into nerves. A mature neuron-specific fluorescent reporter (GFP-Synapsin) was introduced using a lentiviral vector. Thereafter, the culture was switched to adherent culture, and 10 μM each of a low-molecular-weight compound library (Sigma Aldrich) was added to the neuronal differentiation medium and cultured for 17 days (see FIG. 1-A). The fluorescence intensity of GFP-Synapsin was quantified using an imaging cytometer (In cell analyzer). Using astrocyte conditioned medium (ACM), which promotes neuronal differentiation, as a control, compounds exhibiting fluorescence intensity values closer to each other were screened (FIGS. 1 and 2).

(2) The compound that exhibited the highest fluorescence intensity among the selected compounds was KU-60019, an inhibitor of ATM, which is a regulator of the DNA repair mechanism.

Example 2
The following experiment was performed to confirm the effect of KU-60019 on neuronal differentiation and maturation.
(1) When performing neural differentiation from human iPS cells to dopamine neurons, KU-60019 is added to the neural differentiation medium at 1, 5, and 10 μM and cultured for 10 days to express Nestin, a marker of undifferentiated nervous system. Cells were quantified to evaluate differentiation efficiency (culture conditions are shown in FIG. 3). As a result, the amount of Nestin-positive cells decreased in a KU-60019 concentration-dependent manner (FIGS. 4 and 5). This suggests that KU-60019 has the effect of promoting the differentiation of neural stem cells into nerve cells.

(2) In order to compare the amount of dopaminergic neurons (DAN) differentiated by the same method as in (1), tyrosine hydroxylase (TH)-positive cells, which are specific markers for mature DAN, were quantified. (Fig. 6). As a result, DAN increased in a KU-60019 concentration-dependent manner on day 7 of the culture, suggesting that the efficiency of DAN differentiation was increased.
If the culture is continued up to the 10th day, all progenitor cells differentiate into DAN, and this difference disappears.

Considering that KU-60019 may promote senescence in differentiation from human iPS cells, the following experiment was performed.

(3) Differentiation of DAN was induced using the same method as in (2), collected after 10 days, and the intracellular NAD ⁺ concentration was measured using a colorimetric method (Fig. 7). As a result, the intracellular NAD ⁺ concentration decreased in a KU-60019 concentration-dependent manner. This suggested that KU-60019 promotes cell senescence.

Example 3
As a universal model of cellular senescence, excessive DNA damage and accompanying collapse of the nuclear membrane structure are known. In a study on a progerin-introduced senescence model, it was reported that DAN, which accelerates cellular senescence, causes the collapse of LaminB (nuclear membrane protein) and excessive DNA damage (accumulation of γH2AX) (Cell Stem Cell. 2013). ). Based on this, it was verified whether the same thing is caused in the aging model by the addition of KU-60019.
(1) Human iPS cells were induced to differentiate into DAN using the same method as in Example 2(2), and immunostaining using a LaminB2 antibody was performed on day 7 (FIGS. 8-AC). As a result, it was found that the structure of LaminB1 was destroyed when KU-60019 was added. This suggests that the action of KU-60019 promotes senescence of human iPS cell-derived neurons.

(2) Human iPS cells were induced to differentiate into DAN using the same method as in Example 2 (2), and immunostaining was performed using a γH2AX (Ser139) antibody on day 7 (Fig. 8-D- F). As a result, it was found that γH2AX was accumulated in the nucleus when KU-60019 was added. This suggested that KU-60019 caused excessive DNA damage peculiar to aging models.

Example 4
Since KU-60019 promotes neuronal differentiation and cell senescence from human iPS cells, by adding KU-60019 to the conventional culture method, Parkinson's disease-iPS cells were differentiated from iPS cells. Culture required for phenotype detection in DAN We verified whether the period could be shortened. Among hereditary Parkinson's diseases, a typical one that develops in relatively elderly people (after the 50s) is PARK4 (α-synuclein duplication). Culture durations to phenotype detection were compared.
(1) Differentiation of PARK4-iPS cell-derived DAN was induced using the same method as in Example 2 (2), and then DAN cell loss was analyzed using a TH antibody on days 7 and 10. . As a result, on the 7th day of culture, no significant increase in shedding was observed in both the addition and non-addition of normal control cells. Further culture was continued, and on day 10 of culture, no significant shedding was observed in the KU-60019 non-addition group of PARK4-iPS cells as on day 7, but in the KU-60019 addition group shedding compared to normal control cells It could be confirmed that the number of cells that were treated with this method increased significantly (Fig. 9).

Based on the above results, adding KU-60019 to the culture medium accelerates the senescence of iPS cell-derived neurons, making it possible to detect in a short period of time the late-onset phenotype that was difficult to detect during the conventional culture period. Become.

Example 5
(Observation of senescence-specific nuclear membrane abnormalities in human fibroblasts by addition of KU60019)
When KU60019 was applied to young-derived fibroblasts, senescence-specific nuclear membrane abnormalities (decrease in LaminB1) were observed, showing the same aging phenotype as elderly-derived fibroblasts (LaminB1 (green), Lamin A/C (red)) (Fig. 10A). The ratio of cells showing normal nuclear membrane structure in juvenile-derived fibroblasts was decreased by the action of KU60019 (mean±SEM, n=6, *p<0.01.) (FIG. 10B).
In fibroblasts collected from an elderly person (51 years old), when compared with human fibroblasts collected from a young person (18 years old), a decrease in LaminB1, a nuclear membrane marker, was observed as an aging-specific nuclear membrane abnormality. be done. When KU60019 was added to juvenile-derived fibroblasts for 72 hours, a decrease in LaminB1 was observed, and a senescence-specific phenotype of nuclear envelope collapse was observed (Fig. 10).

Example 6
(Increase in human fibroblast senescence-specific marker SA-Gal by addition of KU60019)
When KU60019 is applied to young-derived fibroblasts, SA-Gal (Senescence associated galactosidase)-positive cells, which are senescence-specific markers, increased to the same level as elderly-derived fibroblasts, and senescence was accelerated (red: SA-Gal blue: nuclear staining) (Fig. 11A). The SA-Gal intensity was similarly increased (mean±SEM, n=6, *p<0.01.) (FIG. 11B).
That is, in the elderly-derived fibroblasts, compared with the young-derived fibroblasts, SA-Gal (Senescence associated galactosidase)-positive cells, which are senescence-specific markers, are increasing, but the young treated with KU60019 In human-derived fibroblasts, SA-Gal-positive cells increased to the level of elderly-derived fibroblasts (Fig. 11).

Example 7
(Senior cell promotion of SHSY5 cells by addition of KU60019)
When KU60019 was applied to SHSY5 cells, a cultured cell line derived from human neuroblastoma, senescence-specific nuclear membrane abnormalities were induced. (Green: LaminB1 red: LaminA/C blue: nuclear staining) (FIG. 12A). Decrease in LaminB1 (LMNB1) mRNA (Fig. 12B), increase in senescence marker p16 mRNA (Fig. 12C), and decrease in senescence marker p21 mRNA were detected by qPCR (mean ± SEM, n = 4, * p < 0 .01.) (Fig. 12D).
That is, even in SHSY5 (immortalized cell line of human nervous system), the addition of KU60019 caused the collapse of the nuclear membrane. In addition, since the expression of LaminB1 (LMNB1) mRNA, which constitutes the nuclear membrane, was also reduced, it was confirmed that cellular senescence was accelerated. It was confirmed that the mRNA expression of senescence-related genes p16 and p21 used in senescence studies of immortalized cells was significantly increased (Fig. 12).

These results indicate that the senescence-promoting action of KU60019 is effective not only on iPS cell-derived neurons, but also on cultured cells in general, including iPS cell-derived neurons.

Example 8
It was verified whether compounds having functions similar to KU60019 have similar effects.
In the same manner as in Example 1, KU60019 (ATM inhibitor), KU55933 (ATM inhibitor), and NU7441 (DNA-PK (a factor related to DNA repair that has a function similar to ATM) inhibitor to human IPS cell-derived neurons ), it was confirmed that only the cells to which KU60019 was added promoted maturation (Fig. 13). From these results, it is speculated that only KU60019 among ATM inhibitors and similar agents has cell maturation-promoting/senescence-inducing activity.

Claims (4)

A maturing senescence-promoting agent for unsenescent nerve cells or fibroblasts containing KU-60019 as an active ingredient, wherein the concentration of KU-60019 in the medium at the time of use is 1 μM to 10 μM . 2. The agent for promoting mature aging according to claim 1 , wherein the unsenescent nerve cells or fibroblasts are unsenescent nerve cells or fibroblasts derived from pluripotent stem cells. A method for promoting mature senescence of nerve cells or fibroblasts, which comprises culturing unsenescent nerve cells or fibroblasts in a medium containing 1 μM to 10 μM of KU-60019. 4. The method for promoting mature aging according to claim 3 , wherein the unsenescent nerve cells or fibroblasts are pluripotent stem cell-derived unsenescent nerve cells or fibroblasts.

Images