A parkinson's disease cell model with mitochondrial damage and a construction method and application thereof

Abstract

The application discloses a Parkinson's disease cell model with mitochondrial damage, a construction method and application thereof. The construction method utilizes a target virus containing a CRISPR / Cas9 system to infect dopaminergic neuron cells, can precisely knock out a gene fragment coding mitochondrial transcription factor A in a nucleus gene of the dopaminergic neuron cells, makes the dopaminergic neuron cells reduce expression of TFAM protein, causes mitochondrial dysfunction, and further forms the Parkinson's disease cell model with mitochondrial damage. The mitochondrial damage feature of the Parkinson's disease cell model provided by the application is hereditary, therefore, after CRISPR / Cas9 gene editing is performed on primary cells, the edited primary cells are cultured to obtain a sufficient number of cell models for subsequent experiments and research. Compared with a method of exposing to rotenone, the method does not need to re-model each time the Parkinson's disease cell model is used, and the subcultured cells can be directly taken.

Description

Technical Field

[0001] This invention belongs to the field of cell model construction, and relates to a Parkinson's disease cell model with mitochondrial damage, its construction method, and its applications. Background Technology

[0002] Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra of the midbrain and the presence of protein inclusions in the Lewy body. Patients typically exhibit a range of motor disturbances, including resting tremor, bradykinesia, postural instability, and rigidity in the neck, trunk, and limbs. With the increasing aging of the global population, the number of people with Parkinson's disease is rapidly increasing.

[0003] Recent studies have revealed the crucial role of epigenomic alterations in the neuropathology and etiology of Parkinson's disease. Furthermore, existing research indicates that mitochondrial dysfunction plays a vital role in the pathogenesis of Parkinson's disease. For example, the neurotoxic pesticide rotenone can induce core histone acetylation in cultured dopaminergic neurons, leading to severe mitochondrial dysfunction in dopaminergic neurons exposed to rotenone. Reports indicate elevated levels of core histone acetylation in animal models of Parkinson's disease and in the brains of human patients, suggesting a possible pathological mechanism linking mitochondrial dysfunction and excessive core histone acetylation in Parkinson's disease.

[0004] Although short-term exposure to rotenone can induce mitochondrial damage in neurons, its efficiency is low, making it difficult to rapidly obtain large quantities of suitable cellular resources for research. Therefore, developing a cellular model of mitochondrial damage associated with Parkinson's disease is particularly important. Summary of the Invention

[0005] To address the problems of low efficiency and difficulty in rapidly obtaining large quantities of suitable cell resources for research in existing methods for constructing Parkinson's disease cell models with mitochondrial damage, this invention provides a Parkinson's disease cell model with mitochondrial damage, its construction method, and its applications. This construction method utilizes a virus containing the CRISPR / Cas9 system to infect dopaminergic neurons, precisely knocking out the gene segment encoding mitochondrial transcription factor A (hereinafter referred to as TFAM protein) in dopaminergic neurons (hereinafter referred to as the TFAM gene). This reduces the expression of TFAM protein in dopaminergic neurons, thereby causing mitochondrial dysfunction and forming a Parkinson's disease cell model with mitochondrial damage. The passaged cells of the Parkinson's disease cell model obtained by this method remain Parkinson's disease cell models.

[0006] The technical solution provided by this invention is as follows:

[0007] In a first aspect, the present invention provides a plasmid for inducing mitochondrial damage in cells, characterized in that: the plasmid comprises a CRISPR / Cas9 system;

[0008] The CRISPR / Cas9 system includes a gRNA that targets exon 1 of the TFAM gene.

[0009] Secondly, the present invention provides a virus for causing mitochondrial damage in cells, the virus comprising the aforementioned plasmid.

[0010] Thirdly, the present invention provides a Parkinson's disease cell model with mitochondrial damage, wherein the gene segment encoding TFAM protein is knocked out in the Parkinson's disease cell model.

[0011] Fourthly, the present invention provides a method for constructing a Parkinson's disease cell model with mitochondrial damage, comprising:

[0012] Dopaminergic neurons were infected with a target virus containing the CRISPR / Cas9 system, and the TFAM gene in the dopaminergic neurons was knocked out to obtain a Parkinson's disease cell model with mitochondrial damage.

[0013] The TFAM gene is a gene segment encoding the TFAM protein;

[0014] The CRISPR / Cas9 system includes a gRNA that targets exon 1 of the TFAM gene.

[0015] In some embodiments of the present invention, the method for constructing a Parkinson's disease cell model with mitochondrial damage provided by the present invention further includes:

[0016] The target virus was obtained by packaging the sgRNA and the gene encoding the CAS protein using the virus.

[0017] 293FT cells were infected with a target virus containing the CRISPR / Cas9 system to amplify the target virus.

[0018] In some embodiments of the present invention, the method for constructing a Parkinson's disease cell model with mitochondrial damage provided by the present invention reduces the expression of TFAM protein in dopaminergic neurons by more than 90%.

[0019] In some embodiments of the present invention, the target virus is a lentivirus.

[0020] In some embodiments of the present invention, infecting dopaminergic neurons with a target virus containing a CRISPR / Cas9 system includes: seeding dopaminergic neurons in a complete culture medium, adding the target virus when the cells reach 90% contact fusion, co-culturing for 24 hours, replacing the complete culture medium with a selection medium, and screening out cells that have successfully expressed the transfected virus.

[0021] In some embodiments of the present invention, the target virus contains the pac gene, and the selection culture medium contains puromycin.

[0022] In some embodiments of the present invention, the dopaminergic neurons are N27 cell lines.

[0023] Fifthly, the present invention provides an application of a Parkinson's disease cell model with mitochondrial damage in Parkinson's disease research, wherein the Parkinson's disease research is for non-disease diagnosis and treatment purposes.

[0024] In a sixth aspect, the present invention provides the use of a Parkinson's disease cell model with mitochondrial damage in screening drugs for the prevention and / or treatment of Parkinson's disease.

[0025] Compared with the prior art, the present invention has at least the following beneficial effects:

[0026] The mitochondrial damage characteristics of the Parkinson's disease cell model provided by this invention are hereditary. Therefore, this invention involves CRISPR / Cas9 gene editing of primary cells, followed by culturing the edited primary cells to obtain a sufficient number of cells for subsequent experiments and research. Compared to the rotenone exposure method, this method eliminates the need for re-establishing the Parkinson's disease cell model each time it is used, allowing direct use of passaged cells. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 A: Results of Western blotting experiment; A shows the difference in TFAM protein expression levels between the control and treatment groups as shown by Western blotting experiment; B is a statistical graph of A.

[0029] Figure 2 The experimental results show that the gene editing of this invention resulted in increased mitochondrial membrane depolarization, increased mitochondrial circulation, and mitochondrial structural damage in Parkinson's disease-related mitochondrial injury cells.

[0030] Figure 3 Experimental results show that the gene-edited cells of this invention exhibit reduced mitochondrial basal respiration rate, decreased ATP production, and increased mitochondrial reactive oxygen species in Parkinson's disease-associated mitochondrial damage.

[0031] Figure 4 Experimental results show that the gene-edited cells of this invention significantly enhanced the signaling of the core histone H3K27ac in Parkinson's disease-associated mitochondrial damage.

[0032] Figure 5 Experimental results show that the gene-edited Parkinson's disease-associated mitochondrial damage cells of this invention exhibit transcriptomic changes similar to those of dopaminergic neurons exposed to rotenone. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0034] For simplicity, this invention only explicitly discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form a range not explicitly stated; and any lower limit can be combined with other lower limits to form a range not explicitly stated; similarly, any upper limit can be combined with any other upper limit to form a range not explicitly stated. Furthermore, although not explicitly stated, every point or individual value between the endpoints of the range is included within that range. Therefore, each point or individual value can be used as its own lower or upper limit and combined with any other point or individual value, or combined with other lower or upper limits, to form a range not explicitly stated.

[0035] It should be noted that, in the description of this invention, unless otherwise stated, "above" and "below" include the stated number, and "multiple" in "one or more" means two or more. Relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0036] In the description of this invention, the terms "any embodiment / mode," "one embodiment / mode," "some embodiments / modes," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment / mode or example, which are included in at least one embodiment / mode or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment / mode or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments / modes or examples. Furthermore, without contradiction, those skilled in the art can combine and integrate the different embodiments / modes or examples described in this specification, as well as the features of different embodiments / modes or examples.

[0037] The above description of the invention is not intended to describe every disclosed embodiment or implementation of the invention. Exemplary embodiments are described in more detail below. These embodiments can be used in various combinations. In each example, the listing is merely representative and should not be construed as exhaustive.

[0038] Unless otherwise specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art in the relevant field.

[0039] plasmid:

[0040] The present invention provides a plasmid for inducing mitochondrial damage in cells, the plasmid comprising a CRISPR / Cas9 system; the CRISPR / Cas9 system comprising a gRNA targeting exon 1 of the TFAM gene.

[0041] After the plasmid is transfected into the cell, the gRNA can target and bind to exon 1 of the TFAM gene, thereby knocking out the TFAM gene in the cell and reducing the expression of TFAM protein, thus causing mitochondrial damage.

[0042] Virus:

[0043] The present invention provides a virus for causing mitochondrial damage in cells, the virus containing the aforementioned plasmid.

[0044] The virus contains the aforementioned plasmid. After transfecting the virus into cells, the plasmid can be released. The gRNA of the CRISPR / Cas9 system in the plasmid targets and binds to exon 1 of the TFAM gene. The Cas9 protein precisely knocks out the TFAM gene in the cell, reducing the expression of the TFAM protein, thereby causing mitochondrial damage in the cell.

[0045] Parkinson's disease cell model:

[0046] In the Parkinson's disease cell model with mitochondrial damage provided by this invention, the gene segment encoding TFAM protein was knocked out.

[0047] When the TFAM gene in a cell is knocked out, the expression of the TFAM protein decreases, mitochondrial dysfunction occurs, and the cell becomes a cell model of Parkinson's disease.

[0048] Cell model construction methods:

[0049] The method for constructing a Parkinson's disease cell model with mitochondrial damage provided by the present invention includes:

[0050] Dopaminergic neurons were infected with a target virus containing the CRISPR / Cas9 system, and the TFAM gene in the dopaminergic neurons was knocked out to obtain a Parkinson's disease cell model with mitochondrial damage.

[0051] The TFAM gene is a gene segment encoding the TFAM protein;

[0052] The CRISPR / Cas9 system includes a gRNA that targets exon 1 of the TFAM gene.

[0053] The TFAM gene plays a crucial role in regulating mitochondrial function in mammals. For example, interfering with TFAM gene expression can lead to mitochondrial DNA depletion, loss of mitochondrial transcripts, loss of mitochondrial DNA-encoded peptides, and severe respiratory chain defects, ultimately resulting in reduced mitochondrial ATP production.

[0054] This invention utilizes a target virus carrying a CRISPR / Cas9 system to precisely knock out the TFAM gene in dopaminergic neurons. The resulting Parkinson's disease cell model with mitochondrial damage (TFAM-KO N27) can stably and significantly reduce the expression of TFAM protein, and its mitochondrial function is significantly impaired. At the same time, the H3K27ac signal of the core histone H3 in the cell nucleus is significantly enhanced, which is consistent with the mitochondrial damage phenotype of dopaminergic neurons caused by traditional environmental toxin exposure—rotenone exposure.

[0055] This invention provides a novel method for constructing a Parkinson's disease cell model, and the obtained Parkinson's disease cell model is a high-purity, stable, and genetically inherited cell model, providing a reliable experimental platform for studying the molecular mechanisms of mitochondrial damage and histone acetylation in Parkinson's disease.

[0056] This invention involves CRISPR / Cas9 gene editing of primary cells, followed by passage culture of the edited primary cells to obtain a sufficient number of cell models for subsequent experiments and research. Compared to the rotenone exposure method, this method eliminates the need for re-establishing the Parkinson's disease cell model each time it is used, allowing direct use of passaged cells.

[0057] The CRISPR / Cas9 system in the target virus of this invention contains sgRNA that directly and specifically targets exon 1 of TFAM, thus enabling rapid and targeted knockout of the TFAM gene.

[0058] In some embodiments of the present invention, the method for constructing a Parkinson's disease cell model with mitochondrial damage provided by the present invention further includes:

[0059] The target virus was obtained by packaging the sgRNA and the gene encoding the CAS protein using the virus.

[0060] 293FT cells were infected with a target virus containing the CRISPR / Cas9 system to amplify the target virus.

[0061] 293FT cells are a rapidly growing clonal isolate, making them suitable for large-scale bioreactor culture, thereby enabling large-scale production of lentiviruses. After obtaining the target virus, this invention transfects the target virus into 293FT cells and uses 293FT cells to amplify the target virus in large quantities, which can obtain a sufficient amount of target virus in a short time.

[0062] In some embodiments of the present invention, the method for constructing a Parkinson's disease cell model of mitochondrial damage provided by the present invention reduces the expression of TFAM protein in dopaminergic neurons by more than 90%, thereby restricting mitochondrial function and causing mitochondrial dysfunction.

[0063] In some embodiments of the present invention, the target virus is a lentivirus. Lentiviral vectors contain the VSV-G envelope protein, exhibiting broad host cell affinity and capable of transducing almost all mammalian cells, including dividing cells, non-dividing cells, and primary cells. Compared to traditional plasmid transfection, lentiviral transduction allows for more uniform transfer of exogenous genes into target cells. Furthermore, the CRISPR / Cas9 system packaged with lentiviruses does not require a virus-grade laboratory environment, significantly reducing the environmental requirements for gene editing experiments.

[0064] In some embodiments of the present invention, infecting dopaminergic neurons with a target virus containing the CRISPR / Cas9 system includes: seeding dopaminergic neurons in complete culture medium; adding the target virus when cell contact fusion reaches 90%; co-culturing for 24 hours; and then replacing the complete culture medium with selection medium to screen for cells that have successfully expressed the transfected virus. This method effectively introduces the CRISPR / Cas9 system into dopaminergic neurons, achieving knockout of the TFAM gene.

[0065] In some embodiments of the present invention, the target virus contains the pac gene, and the selection medium contains puromycin. The selection medium containing puromycin can kill dopaminergic neurons that do not contain the pac gene, thereby screening out dopaminergic neurons successfully transfected with the target virus containing the pac gene.

[0066] In some embodiments of this invention, the dopaminergic neurons are N27 cell lines. The N27 cell line is a cell line derived from dopaminergic neurons in the rat embryonic mesencephalon and is widely used in Parkinson's disease research, where it can be used to study neurotoxicity, oxidative stress, neurodegeneration, and other molecular pathways. This invention utilizes the N27 cell line to construct a Parkinson's disease cell model, thereby obtaining a cell model that more closely resembles Parkinson's disease.

[0067] Parkinson's disease cell model:

[0068] The Parkinson's disease cell model with mitochondrial damage provided by this invention is obtained through the aforementioned method for constructing a Parkinson's disease cell model with mitochondrial damage. This cell model exhibits increased mitochondrial membrane depolarization, increased mitochondrial circulation, and mitochondrial structural damage; decreased mitochondrial basal respiration rate, reduced ATP production, and increased mitochondrial reactive oxygen species; significantly enhanced H3K27ac signaling of the core histone H3 in the cell nucleus; and transcriptomic alterations similar to those observed in rotenone-exposed dopaminergic neurons. This cell model displays a typical mitochondrial damage phenotype and high levels of core histone hyperacetylation (H3K27ac), and can serve as a cell model for in vitro research on epigenetic molecular mechanisms related to Parkinson's disease. The passaged cells of this Parkinson's disease cell model remain the same as those used in other Parkinson's disease cell models; once properly preserved, cells can be harvested for each passage.

[0069] application:

[0070] The mitochondrial damage-based Parkinson's disease cell model provided by this invention has applications in Parkinson's disease research for purposes other than disease diagnosis and treatment. For example, it can be used to study the pathological mechanisms of Parkinson's disease and the link between dopaminergic neuronal mitochondrial dysfunction and core histone hyperacetylation in Parkinson's disease.

[0071] Furthermore, this invention also provides the application of a Parkinson's disease cell model with mitochondrial damage in screening drugs for the prevention and / or treatment of Parkinson's disease. Those skilled in the art can utilize this Parkinson's disease cell model for in vitro screening of new drugs.

[0072] Example 1: Cell model construction

[0073] 1. Constructing CRISPR / Cas9 system plasmids

[0074] First, a CRISPR / Cas9 system plasmid targeting the transcription factor TFAM sequence was constructed. The gRNA of this plasmid is a nucleotide sequence that can directly target exon 1 of TFAM.

[0075] 2. Packaging CRISPR / Cas9 TFAM-KO lentivirus using 293FT cells

[0076] Plasmids were transfected into 293FT cells using the Mission Lentiviral Packaging Mix kit. Lentiviral was collected 48 hours after vector transfection, and the lentivirus was further concentrated and frozen at -80°C for later use.

[0077] 3. Infection of dopaminergic neuronal cells N27

[0078] The density of dopaminergic N27 cells was adjusted to 1×102 6 ~5×106 Cells were seeded at / mL in culture containers. When cell contact confluence reached 90%, they were infected with CRISPR / Cas9 TFAM-KO lentivirus at a multiplicity of infection (MOI) of 100 and cultured in complete medium. After 24 hours of culture, the complete medium was replaced with selection medium (containing 50 μg / mL puromycin) to obtain a Parkinson's disease cell model with mitochondrial damage (hereinafter referred to as TFAM-KO N27 cells).

[0079] Example 2: Cell Model Validation

[0080] 1. The JC-1 experimental method was used to detect the cell membrane polarization of TFAM-KO N27 cells, and it was found that the mitochondrial membrane depolarization was increased.

[0081] 2. The MitoTracker probe staining method was used to detect the mitochondrial circulation function in TFAM-KO N27 cells. The results showed increased mitochondrial circulation and mitochondrial structural damage.

[0082] 3. The ROS content in mitochondria of TFAM-KO N27 cells was detected by the MitoSox assay. The results showed that mitochondrial ROS increased significantly, indicating an increase in mitochondrial reactive oxygen species.

[0083] 4. Western blotting experiment

[0084] Histones from rotenone-exposed and TFAM-KO N27 cells were enriched using a specific antibody against H3K27ac. Western blotting results showed that, similar to rotenone-exposed N27 cells, the H3K27ac signal of the core histone H3 in the nucleus of TFAM-KO N27 cells was significantly enhanced.

[0085] 5. RNA-seq sequencing analysis

[0086] RNA-seq sequencing was performed on TFAM-KO N27 cells and N27 cells exposed to rotenone, respectively. Differentially expressed genes (DEGs) and gene ontology (GO) clustering analyses were then performed on the RNA-seq results. The results revealed similar transcriptional profiles and altered expression of genes regulating mitochondrial biosynthesis. Therefore, TFAM-KO N27 cells exhibit the same mitochondrial damage phenotype as rotenone-exposed N27 cells.

[0087] In summary, the Parkinson's disease cell model with mitochondrial damage provided by this invention significantly reduces the expression of TFAM protein, exhibits a typical mitochondrial damage phenotype and a high level of core histone hyperacetylation (H3K27ac), and can be used as a cell model for in vitro research on epigenetic molecular mechanisms related to Parkinson's disease.

[0088] The above are merely specific embodiments of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A plasmid for inducing mitochondrial damage in cells, characterized in that: The plasmid includes a CRISPR / Cas9 system; The CRISPR / Cas9 system includes a gRNA that targets exon 1 of the TFAM gene.

2. A virus for inducing mitochondrial damage in cells, characterized in that: The virus contains the plasmid as described in claim 1.

3. A Parkinson's disease cell model with mitochondrial damage, characterized in that, In the Parkinson's disease cell model, the gene segment encoding the TFAM protein was knocked out.

4. A method for constructing a Parkinson's disease cell model with mitochondrial damage, characterized in that, include: Dopaminergic neurons were infected with a target virus containing the CRISPR / Cas9 system, and the TFAM gene in the dopaminergic neurons was knocked out to obtain a Parkinson's disease cell model with mitochondrial damage. The TFAM gene is a gene segment encoding the TFAM protein; The CRISPR / Cas9 system includes a gRNA that targets exon 1 of the TFAM gene.

5. The method for constructing a Parkinson's disease cell model with mitochondrial damage according to claim 4, characterized in that: The method for constructing the Parkinson's disease cell model of mitochondrial damage also includes: The target virus was obtained by packaging the sgRNA and the gene encoding the CAS protein using the virus. 293FT cells were infected with a target virus containing the CRISPR / Cas9 system to amplify the target virus.

6. The method for constructing a Parkinson's disease cell model with mitochondrial damage according to claim 4, characterized in that: The target virus is a lentivirus; and / or, The dopaminergic neurons are N27 cell lines.

7. The method for constructing a Parkinson's disease cell model with mitochondrial damage according to claim 4, characterized in that: Infection of dopaminergic neurons with targeted viruses containing the CRISPR / Cas9 system includes: Dopaminergic neurons were seeded in complete culture medium. When the cells reached 90% contact fusion, the target virus was added and co-cultured for 24 hours. Then, the complete culture medium was replaced with selection medium to screen out cells that successfully expressed the transfected virus.

8. The method for constructing a Parkinson's disease cell model with mitochondrial damage according to claim 7, characterized in that: The target virus contains the pac gene, and the selection medium contains puromycin.

9. The application of the Parkinson's disease cell model of mitochondrial damage as described in claim 3 in Parkinson's disease research, wherein the application is for purposes other than disease diagnosis and treatment.

10. The use of the Parkinson's disease cell model of mitochondrial damage as described in claim 3 in screening drugs for the prevention and / or treatment of Parkinson's disease.

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