Application of OXA1L in autosomal dominant optic atrophy and mitochondrial dysfunction

Abstract

The invention provides application of OXA1L in autosomal dominant optic atrophy and mitochondrial dysfunction, and relates to the technical field of biology. The application comprises application of a substance for supplementing or up-regulating OXA1L in preparing a product for relieving or treating autosomal dominant optic atrophy or application of a substance for supplementing or up-regulating OXA1L in preparing a product for relieving or treating mitochondrial dysfunction caused by AFG3L2 mutation. The invention finds that up-regulation of OXA1L expression can improve mitochondrial dysfunction caused by AFG3L2 mutation, and provides a new strategy for treatment of autosomal dominant optic atrophy.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an application of OXA1L in autosomal dominant optic atrophy and mitochondrial dysfunction. Background Art

[0002] The following statements merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] Autosomal dominant optic atrophy (DOA) is an optic neuropathy caused by degeneration of optic nerve fibers. It is the most common hereditary optic nerve disease with an incidence of about 1:12,000 to 25,000. Patients suffer from decreased vision in both eyes and even blindness. Due to the lack of effective treatments, it is still an incurable disease in ophthalmology, bringing a huge economic burden to patients and their families. Therefore, it is of great significance to clarify the pathological mechanism of DOA and discover effective and specific therapeutic targets for the treatment of DOA.

[0004] DOA usually develops in infancy and presents a long pathological process. Clinical manifestations include moderate to severe visual loss, binocular optic atrophy, central or paracentral scotoma, and color vision defects. At present, there is a lack of effective measures for the treatment of DOA, and only a few treatment directions have been proposed. The first is gene therapy, which is to restore the wild-type gene by correcting the pathological mutation. RGCs can be targeted with the help of CRISPR-Cas9 tools and viral vectors AAV to trigger the recombination of mutant gene sites. However, this has caused many concerns because this method cannot control the level of transgene expression in retinal ganglion cells, and it is also necessary to solve the problems of haploid insufficiency and dominant negative mutations. Secondly, drug treatments for mitochondrial diseases mainly counteract mitochondrial dysfunction and activate mitochondrial respiration through systemic application of antioxidant drugs or molecules such as AICAR and rapamycin; or through neuroprotection and growth factor therapy, targeted administration of survival proteins to protect RGCs persistently. However, so far these methods have not shown significant protection of the optic nerve. In addition, some studies have proposed to restore normal optic nerves through the regeneration of RGCs. For example, targeted downregulation of PTBP1, a gene encoding RNA polypyrimidine binding protein 1, converts mouse Müller glial cells into retinal ganglion cells; Schwann cells are transplanted into the vitreous to promote the secretion of neurotrophic factors and RGC axon regeneration. However, this may still require further exploration and demonstration.

[0005] AFG3L2 F175As a new mutation that can cause typical DOA symptoms discovered in recent years, it is a variation in the intermembrane domain. Patients have poor vision since childhood, accompanied by progressive aggravation. Due to the lack of effective treatment, it is still an incurable disease in ophthalmology. At present, there is no effective treatment strategy that can target DOA associated with AFG3L2 intermembrane domain mutations.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide a new application of a substance that supplements or upregulates OXA1L, and to provide a new strategy for the treatment of autosomal dominant optic atrophy and mitochondrial dysfunction caused by AFG3L2 mutation.

[0008] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions:

[0009] First, the use of substances that supplement or upregulate OXA1L in (I) or (II):

[0010] (I) Preparation of products for alleviating or treating autosomal dominant optic atrophy;

[0011] (II) Prepare products to alleviate or treat mitochondrial dysfunction caused by AFG3L2 mutation.

[0012] In an optional embodiment, the product further comprises a substance that supplements or upregulates SOD2.

[0013] In a second aspect, a pharmaceutical composition is also provided, which is used to alleviate or treat autosomal dominant optic atrophy; or to alleviate or treat mitochondrial dysfunction caused by AFG3L2 mutation; the pharmaceutical composition comprises a substance that supplements or upregulates OXA1L, and a substance that supplements or upregulates SOD2.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention has found through experiments that OXA1L may be a key protein for AFG3L2 to exert its biological function, and AFG3L2 mutation may cause AFG3L2 to regulate OXA1L. Upregulating the expression of OXA1L and SOD2 in test cells with AFG3L2 mutation can improve mitochondrial dysfunction caused by mutation. It is feasible to use OXA1L and, optionally, SOD2 as therapeutic targets for AFG3L2 mutation-related DOA, providing a new therapeutic strategy for the treatment of DOA and mitochondrial dysfunction caused by AFG3L2 mutation. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 To overexpress AFG3L2 in HEK293T cells WT and AFG3L2 F175S Protein, Western Blot detection of OXA1L protein in HEK293T cells;

[0018] Figure 2 AFG3L2 is overexpressed in HEK293T cells in Example 1 WT and AFG3L2 F175S After protein, proteomic analysis volcano map (AFG3L2 F175S vs AFG3L2 WT );

[0019] Figure 3 For co-transfection of AFG3L2 in HEK293T cell line in Example 2 F175S and OE_OXA1L, and then Western Blot was used to detect the expression of related proteins in cells;

[0020] Figure 4 For co-transfection of AFG3L2 in HEK293T cell line in Example 2 F175S and OE_SOD2, and then Western Blot was used to detect the expression of related proteins in cells;

[0021] Figure 5 The results of detecting ROS levels in the cytoplasm using the fluorescent probe DCFH-DA reactive oxygen species detection kit after successful plasmid transfection of each experimental group of HEK293T cells in Example 2, the scale bar is 100 μm;

[0022] Figure 6 The results of the JC-1 fluorescent probe mitochondrial membrane potential detection kit detecting the cell membrane potential level after the plasmids of each experimental group of HEK293T cells were successfully transfected in Example 2. The scale bar is 100 μm.

[0023] Figure 7 After the plasmids of each experimental group of HEK293T cells were successfully transfected in Example 2, MitoSOX TM The results of measuring mitochondrial ROS levels with Red reagent, the scale bar is 20 μm;

[0024] Figure 8 The expression of related proteins in the cells was detected by Western Blot after the immortalized fibroblasts of DOA patients were transfected with OE_SOD2 plasmid (a) and OE_OXA1L plasmid (b) in Example 2;

[0025] Fig. 9 The results of detecting ROS levels in the cytoplasm using the fluorescent probe DCFH-DA reactive oxygen species detection kit after successful plasmid transfection of each experimental group of iFB cells in Example 2, the scale bar is 100 μm;

[0026] Fig.10 After successful plasmid transfection of each experimental group of iFB cells in Example 2, MitoSOX TM The results of measuring mitochondrial ROS levels using Red reagent, the scale bar is 100 μm;

[0027] Fig.11 The results of the JC-1 fluorescent probe mitochondrial membrane potential detection kit detecting the cell membrane potential level after the plasmids of each experimental group of iFB cells were successfully transfected in Example 2. The scale bar is 100 μm.

[0028] Fig.12 The mitochondrial morphology of iFB cells after successful plasmid transfection of each experimental group of iFB cells in Example 2, the scale bar is 10 μm. DETAILED DESCRIPTION

[0029] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references, and the laboratory procedures and techniques thereof are those well known and commonly used in the art.

[0031] In a first aspect, a use of a substance for supplementing or upregulating OXA1L in (I) or (II) is provided:

[0032] (I) Preparation of products for alleviating or treating autosomal dominant optic atrophy;

[0033] (II) Prepare products to alleviate or treat mitochondrial dysfunction caused by AFG3L2 mutation.

[0034] OXA1L (OXA1L mitochondrial inner membrane protein) is located in the mitochondrial inner membrane and is essential for the translocation of the N-terminal tail of cytochrome c oxidase subunit II and is involved in the assembly of the cytochrome c oxidase and ATPase complexes of the mitochondrial respiratory chain.

[0035] In an optional embodiment, the autosomal dominant optic atrophy is caused by a mutation in AFG3L2 (AFG3like matrixAAA peptidase subunit 2). AFG3L2 is a proteolytic enzyme that plays a major role in the m-AAA (membrane-Anchored AAA, m-AAA) proteasome. AFG3L2 self-assembles in the inner membrane of mitochondria and relies on ATP for energy supply to maintain mitochondrial protein stability and efficient oxidative phosphorylation activity.

[0036] In alternative embodiments, the AFG3L2 mutation that causes autosomal dominant optic atrophy comprises a mutation in the AFG3L2 intermembrane domain.

[0037] In an alternative embodiment, the AFG3L2 mutation causing autosomal dominant optic atrophy comprises AFG3L2 F175 Mutation, AFG3L2 F175 This indicates that the F (phenylalanine) at position 175 of AFG3L2 has mutated.

[0038] In an alternative embodiment, AFG3L2 causes autosomal dominant optic atrophy F175 Mutations include AFG3L2 F175S Mutation, AFG3L2 F175S This indicates that the F (phenylalanine) at position 175 of AFG3L2 was mutated to S (serine).

[0039] In an optional embodiment, the alleviation or treatment of autosomal dominant optic atrophy includes one or more of reducing cellular ROS levels, reducing mitochondrial ROS levels and increasing mitochondrial membrane potential.

[0040] In an optional embodiment, the mitochondrial dysfunction caused by the AFG3L2 mutation includes mitochondrial dysfunction caused by a mutation in the intermembrane domain of AFG3L2.

[0041] In an optional embodiment, the mitochondrial dysfunction caused by the AFG3L2 mutation includes AFG3L2 F175 Mutation-induced mitochondrial dysfunction.

[0042] In an optional embodiment, the mitochondrial dysfunction caused by the AFG3L2 mutation includes AFG3L2 F175S Mutation-induced mitochondrial dysfunction.

[0043] In an optional embodiment, the alleviation or treatment of mitochondrial dysfunction caused by AFG3L2 mutation includes one or more of reducing cellular ROS levels, reducing mitochondrial ROS levels and increasing mitochondrial membrane potential.

[0044] In an optional embodiment, the subject of the product in (I) or (II) includes a mammal or a cell derived from a mammal, and the mammal includes but is not limited to humans, pigs, cattle, horses, monkeys, rats, mice, guinea pigs, sheep, goats or goats. The subject can be a patient with autosomal dominant optic atrophy or a patient with mitochondrial dysfunction caused by AFG3L2 mutation, or a cell derived from any of the above patients, or an animal model or cell model of autosomal dominant optic atrophy or mitochondrial dysfunction caused by AFG3L2 mutation.

[0045] The substance that supplements or upregulates OXA1L can be any substance known in the art that is acceptable and can increase the OXA1L content in a subject, and includes but is not limited to at least one of OXA1L protein, a polynucleotide encoding the OXA1L gene, and an OXA1L agonist, wherein the OXA1L agonist promotes OXA1L gene expression and / or protein synthesis in a subject.

[0046] In an optional embodiment, the product further comprises a substance for supplementing or up-regulating SOD2. The substance for supplementing or up-regulating SOD2 may be any substance known in the art and acceptable to increase the SOD2 content of a subject, and the substance for supplementing or up-regulating SOD2 includes but is not limited to at least one of SOD2 protein, a polynucleotide encoding a SOD2 gene, and a SOD2 agonist, and the SOD2 agonist promotes SOD2 gene expression and / or protein synthesis in a subject.

[0047] OXA1L protein and SOD2 protein include but are not limited to proteins naturally expressed by cells or expressed by cells transfected with polynucleotides encoding OXA1L or SOD2. The above proteins also include artificially modified proteins, including but not limited to polypeptides or proteins after mutation, truncation or fusion with other domains, and polypeptides or proteins that retain the original functions of OXA1L or SOD2 protein.

[0048] As used herein, "polynucleotide" refers to a polymeric form of nucleotides of any length, including ribonucleotides and / or deoxyribonucleotides. Examples of polynucleotides include, but are not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases. The polynucleotide encodes OXA1L or SOD2, optionally encoding a sense strand or an antisense strand. The polynucleotide may be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "polynucleotide" and "nucleic acid" are used interchangeably herein.

[0049] In an optional embodiment, the substance for supplementing or upregulating OXA1L comprises a vector carrying the gene encoding OXA1L.

[0050] In an optional embodiment, the substance for supplementing or upregulating SOD2 includes a vector carrying the gene encoding SOD2.

[0051] The "vector" herein includes, but is not limited to, plasmids, minicircle plasmids, episomal plasmids, phagemids, cosmids, artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC), bacteriophages such as λ phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses.

[0052] In an optional embodiment, the vector contains regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0053] In a second aspect, a pharmaceutical composition is also provided, which is used to alleviate or treat autosomal dominant optic atrophy; or to alleviate or treat mitochondrial dysfunction caused by AFG3L2 mutation; the pharmaceutical composition comprises a substance that supplements or upregulates OXA1L, and a substance that supplements or upregulates SOD2.

[0054] In an optional embodiment, the substance that supplements or upregulates OXA1L includes at least one of OXA1L protein, a polynucleotide encoding the OXA1L gene, and an OXA1L agonist, and the OXA1L agonist promotes OXA1L gene expression and / or protein synthesis in the subject.

[0055] In an optional embodiment, the substance for supplementing or upregulating OXA1L comprises a vector carrying the gene encoding OXA1L.

[0056] In an optional embodiment, the substance that supplements or upregulates SOD2 includes at least one of a SOD2 protein, a polynucleotide encoding a SOD2 gene, and a SOD2 agonist, and the SOD2 agonist promotes SOD2 gene expression and / or protein synthesis in the subject.

[0057] In an optional embodiment, the substance for supplementing or upregulating SOD2 includes a vector carrying the gene encoding SOD2.

[0058] In an optional embodiment, the pharmaceutical composition further contains optional pharmaceutically acceptable excipients. Wherein "optional" means that the pharmaceutical composition may or may not include excipients, and as long as it is an excipient acceptable in the art for drug preparation, those skilled in the art can proceed according to methods well known in the art and recorded in various general and more specific teaching materials, references, process manuals, product descriptions and standard documents, etc., and the present invention does not limit this. Specific excipients include, but are not limited to, one or more of solvents, solubilizers, cosolvents, emulsifiers, colorants, osmotic pressure regulators, stabilizers, antibacterial agents, suspending agents, antioxidants, chelating agents, pH regulators, adsorbents and surfactants.

[0059] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0060] Example 1

[0061] Target screening

[0062] 1. Culture of HEK293T cells

[0063] HEK293T cell line was purchased from the American Type Culture Collection (ATCC) and STR identification was performed. The cells were grown in high-glucose DMEM medium supplemented with 10% fetal bovine serum and 100U penicillin / streptomycin, and cultured in a cell culture incubator at 37°C with 5% CO2. The cells were passaged when they grew to 80% to 90% density.

[0064] 2. Plasmid construction, extraction and transfection

[0065] The CDS sequences of wild-type AFG3L2 (NM_006796.3) and C.524T>C (p.F175S) containing a C-terminal Flag tag were cloned into the eukaryotic expression vector pcDNA3.1 to construct AFG3L2 WTand AFG3L2 F175S After glycerol bacteria activation and plasmid extraction, AFG3L2 was obtained. WT and AFG3L2 F175S Plasmid.

[0066] The cells were plated at 3 × 10 5 Density was plated in 6-well plates, and cells were transfected according to the operating instructions after 70% of the cells adhered to the wall. Transfection reagent was prepared for each well: A solution (125 μL Opti-DMEM TM Culture medium, 7.5 μL Lipofectamine TM 3000 reagent), solution B (125 μL Opti-DMEM TM Culture medium, 5 μL P3000 TM Reagent, 2.5μg plasmid), add solution B to solution A and mix well, incubate at room temperature for 10-15 minutes. Aspirate the old culture medium, add fresh basal culture medium, drop the plasmid-lipid complex into the cell culture plate, and add FBS to 5-10% concentration according to the cell state after 5-6 hours. According to the above transfection method, AFG3L2 was transfected into HEK293T cells respectively. WT Plasmid, AFG3L2 F175S Plasmid and empty vector plasmid pcDNA3.1.

[0067] 3. Identification of HEK293T cell line model

[0068] Protein expression was detected by Western blot. Figure 1 As shown, compared with the no-load group, AFG3L2 WT and AFG3L2 F175S Plasmid-transfected HEK293T cells showed a decreased expression level of OXA1L protein; and AFG3L2 F175S The OXA1L protein level in the transfected group decreased more significantly and was significantly lower than that in the AFG3L2 WT Plasmid transfection group.

[0069] 4. Mass spectrometry analysis of HEK 293T model organisms

[0070] 1. Biological Mass Spectrometry Sample Preparation

[0071] Add 500 μL of urea lysis buffer (8M urea, 50mM NH4HCO3, 1× Roche protease inhibitor) to the sample, lyse at room temperature for 5 minutes, then sonicate on ice (energy 35%, supersonication for 3 seconds, stop for 3 seconds, sonication for 2 minutes), centrifuge at 14000g for 10 minutes, take the supernatant and repeat centrifugation. For BCA determination of protein concentration, take 100 μg of protein from each sample, add DTT (final concentration 10mM), incubate at 37°C for 1 hour for reduction, add IAA (final concentration 40mM), incubate at room temperature for 1 hour in the dark for alkylation. Add 300 μL of 50mM NH4HCO3 to balance the 10kDa ultrafiltration tube twice (centrifuge at 14000g for 15 minutes), add 100 μg of the reduced and alkylated sample, centrifuge at 14000g for 15 minutes at room temperature, add 300 μL of 50mM NH4HCO3 to wash 3 times, and replace the collection tube. Add 80μL 50mMNH4HCO3 and 3μg pancreatin, incubate at 37℃ for 12-16 hours. The next day, centrifuge at 4℃ 14000g for 20 minutes, add 100μL H2O to rinse twice, add 1% formic acid to terminate enzyme cleavage, and evaporate to dryness at 60℃ in a vacuum. Resuspend the sample with 30μL 0.1% formic acid water and measure the concentration with nanodrop. Take 10μg sample for high pH reverse phase column fractionation. Prepare fractionation reagents: Buffer A is 100% CAN (acetonitrile), Buffer B is 0.1% FA (formic acid), and Elute buffer is a mixture of triethylamine and ACN at different concentrations. Fill the C18 membrane with a 200μL pipette tip, dissolve 30mg high pH resistant C18 filler with Buffer A, add it to the fractionation column, and centrifuge at 4℃ 3000g for 2 minutes. Wash the column with Buffer A and Buffer B respectively and keep it for later use. Load the peptide fragments after enzyme digestion into the fractionation column, repeat loading 3 to 5 times, wash 3 times with 200 μL Buffer B, add different concentrations of Elute buffer for gradient elution, combine 6% and 35% fractions, and take 3 μg of peptide fragments from each fraction for DDA mode mass spectrometry analysis.

[0072] 2. Mass Spectrometry Data Analysis

[0073] The proteinpilot software (version 5.0.1) was used to search the database of the raw data collected in the DDA mode. The enzyme digestion method was trypsin, the database was the Uniprot human database (containing 20368 annotated proteins, released 2020.03), and the unused protScore was > 0.05. The search results were imported into the SWATH software (version 2.0) to quantify the data collected by DIA. The quantitative parameters were set as follows: 6 peptides were selected for each protein, 6 transitions were selected for each peptide, peptide confidence was set to 99%, FDR was set to 1%, modified peptides were excluded, the peak extraction window was 10 minutes, the mass deviation was within 50ppm, 2 endogenous peptides were selected every 10 minutes for retention time correction, and the peak area was output as the quantitative value. The protein expression data were log2 transformed to satisfy the normal distribution, and normalized using the normalize.median function of the R language preprocessCore package to remove proteins without gene names. The t.test function of the R language stats package was used to perform differential analysis of protein expression, and proteins with a P value < 0.05 and a change fold > 1.5 were screened as differential proteins.

[0074] 3. Mass spectrometry analysis results and verification

[0075] Proteomic analysis revealed that OXA1L was a differentially expressed protein in the WT / Vehicle group, MT / Vehicle group, and WT / MT group (WT indicated transfection with AFG3L2 WT Plasmid group, MT represents transfection with AFG3L2 F175S Plasmid group, Vehicle means empty plasmid transfection group), and the expression levels were down-regulated in the three control groups. Compared with the WT group, the expression of OXA1L in the MT group was down-regulated by 0.78 times ( Figure 2 ), which is the protein with the largest down-regulation fold. The above results indicate that OXA1L may be a key protein for AFG3L2 to exert its biological function, and the F175S mutation may impair the function of AFG3L2 in regulating OXA1L.

[0076] The above experimental results indicate that OXA1L is a F175S Mutations lead to key proteins that cause mitochondrial dysfunction. OXA1L is located in the mitochondrial membrane and is a protein related to mitochondrial function. Superoxide dismutase (SOD) can convert superoxide free radicals into hydrogen peroxide and oxygen. SOD2 can improve mitochondrial dysfunction, so subsequent experiments will also use SOD2 as a therapeutic target for DOA.

[0077] Example 2

[0078] Target validation of OXA1L and SOD2

[0079] 1. Plasmid construction, extraction and transfection

[0080] The CDS sequence of wild-type OXA1L (NM_005015.5) containing an N-terminal Flag tag was cloned into the eukaryotic expression vector pcDNA3.1 to construct the OXA1L plasmid (OE_OXA1L), which was verified by Sanger sequencing. The CDS sequence of wild-type SOD2 (NM_005891.3) containing a C-terminal Flag tag was cloned into the eukaryotic expression vector pcDNA3.1 to construct the SOD2 plasmid (OE_SOD2), which was verified by Sanger sequencing. After glycerol bacteria activation and plasmid extraction, the OXA1L plasmid and SOD2 plasmid were obtained.

[0081] 2. Verify the therapeutic effect of targeting OXA1L and SOD2 in HEK293T cell lines.

[0082] 1. The OXA1L plasmid and SOD2 plasmid prepared in step 1 and the AFG3L2 plasmid prepared in Example 1 were WT and AFG3L2 F175S The plasmid and the empty plasmid pcDNA3.1 were transfected into HEK293T cells according to the experimental groups shown in Table 1, and the transfection method was the same as in Example 1.

[0083] Table 1

[0084]

[0085] CCCP is an inhibitor of oxidative phosphorylation and an uncoupler that can induce mitochondrial autophagy.

[0086] 2. Mitochondrial protein extraction

[0087] Same as in Example 1. Protein expression was detected by Western blot.

[0088] 3. Protein sample preparation

[0089] Same as in Example 1. Protein expression was detected by Western blot.

[0090] 4. ROS (Reactive Oxygen Species) Detection

[0091] The fluorescent probe DCFH-DA reactive oxygen species detection kit was used to detect the level of ROS in the cytoplasm. DCFH-DA was diluted to 10 μM in serum-free medium at 1:1000, and the cells were cultured in 6-well plates. 1 mL of diluted DCFH-DA was added to each well and incubated at 37°C for 20 minutes. The cells were washed 3 times with serum-free medium (normal temperature, 50 rpm, 3 minutes each time), and the cells were observed under a fluorescence microscope (Ex / Em=488 / 525 nm), and the images were analyzed using ImageJ software.

[0092] Using MitoSOX TM Red reagent was used to measure mitochondrial ROS, and MitoSOX TM The Red reagent was diluted to 5 μM, the cells were incubated at 37°C for 10 minutes, the cells were washed three times with warm buffer (50 rpm, 3 minutes each time), and the cells were observed using a fluorescence microscope.

[0093] 5. MMP (mitochondrial membrane potential) detection

[0094] Mitochondrial membrane potential was measured using the JC-1 fluorescent probe mitochondrial membrane potential detection kit. Cells were cultured in a 6-well plate to a concentration of 70-80%, JC-1 was diluted by adding 8 mL of ultrapure water per 50 μL of JC-1 (200×), and 2 mL of JC-1 staining buffer (5×) was added to prepare the JC-1 staining working solution. The culture medium was removed, washed with PBS, and 1 mL of culture medium and 1 mL of JC-1 working solution were added, and incubated at 37°C for 20 minutes. The supernatant was removed, and the cells were washed twice with JC-1 staining buffer. The changes in MMP (Δψm) were analyzed under a fluorescence microscope, expressed as the ratio of green fluorescence to red fluorescence, and the images were analyzed using ImageJ software.

[0095] 6. Experimental results:

[0096] Co-transfection of AFG3L2 in HEK293T cell line F175S and OE_OXA1L, Western Blot results are as follows Figure 3 As shown; co-transfection of AFG3L2 in HEK293T cell line F175S and OE_SOD2, Western Blot results are as follows Figure 4 As shown; Figure 3 and Figure 4 This indicated that OE_OXA1L and OE_SOD2 were successfully expressed in HEK293T cell lines.

[0097] The levels of ROS and MMP in the cytoplasm and mitochondria of the control group and OXA1L and SOD2 overexpressing cells were detected. The results showed that the ROS generation in the OXA1L and SOD2 overexpressing group was reduced ( Figure 5 ), increased mitochondrial membrane potential level ( Figure 6 ), mitochondrial ROS generation decreased ( Figure 7 ), suggesting that targeting OXA1L and SOD2 can improve the molecular phenotype of cells.

[0098] 3. Verify the therapeutic effect of targeting OXA1L and SOD2 in immortalized fibroblasts of DOA patients.

[0099] 1. Construction of human skin fibroblast cell line (iFB)

[0100] In obtaining a donor (AFG3L2 F175S After informed consent from patients with DOA with mutations and normal people in the same family, a piece of epithelial tissue of about 1 cm × 1 cm was taken from the inner side of the upper arm of the donor under sterile conditions. After disinfection and incubation, the cells were digested and plated for culture. Immunofluorescence identification was then performed. After the identification was completed, SV40 lentivirus was transfected, and the transfected cells were re-plated and incubated overnight. Puromycin at a concentration of 3 μg / mL (action time 2 days) was added to the culture medium for cell screening, and the screened cells were amplified.

[0101] 2. Super-resolution imaging of mitochondria in iFB cells

[0102] Cells were plated at 1×10 5 The OXA1L plasmid and SOD2 plasmid prepared in step 1 and the control plasmid were transfected into iFB cells according to the experimental groups shown in Table 2. The transfection method was the same as in Example 1.

[0103] Table 2

[0104]

[0105]

[0106] In Table 2, ctrl means no plasmid was transfected, including no empty vector plasmid.

[0107] Staining was performed 24 hours after transfection. Using PKMDR dye: dilute the 250 μM stock solution to a 100 nm working solution, i.e. 1 mL of diluent (basic medium) + 0.4 μL of dye, stain for 10 min, discard the dye, wash twice with PBS and replace with complete medium. Image and take photos under a super-resolution microscope as soon as possible.

[0108] 3. The detection methods of cellular ROS level, mitochondrial ROS level and MMP level are the same as those in the second part of this example.

[0109] 4. Experimental results:

[0110] In DOA patient immortalized fibroblasts (AFG3L2 F175S ) after successfully overexpressing SOD2 and OXA1L ( Figure 8 ), the levels of ROS and MMP in the control group and the SOD2 overexpression group were detected, and the results showed that the generation of ROS in the cytoplasm and mitochondria was reduced ( Fig. 9 and Fig.10 ), the membrane potential level increases ( Fig.11 ); Under mitochondrial super-resolution imaging, the tubular structure of mitochondria in iFB cells of DOA patients transfected with SOD2 plasmid was smooth and complete, with fewer breaks, expanded spheres and fragments ( Fig.12 ). The levels of ROS and MMP in the control group and the OXA1L overexpression group were detected, and the results showed that the generation of ROS in the cytoplasm and mitochondria was reduced ( Fig. 9 and Fig.10 ), the membrane potential level increases ( Fig.11 ).

[0111] The above results prove that overexpression of OXA1L and SOD2 can improve AFG3L2 F175S Mitochondrial dysfunction caused by mutations in AFG3L2 F175S Therapeutic targets for mutation-associated DOA are feasible.

[0112] The method for preparing mitochondrial protein for Western blot detection in the above embodiment is as follows:

[0113] 24 hours after plasmid transfection, wash the cells once with PBS, digest the cells with trypsin cell digestion solution (100-200g, centrifuge at room temperature for 5-10 minutes), and collect the cells. Gently resuspend the cells with pre-cooled PBS (600g, centrifuge at 4℃ for 5 minutes to precipitate the cells, and discard the supernatant). Add 1-2.5mL of mitochondrial isolation reagent with PMSF added to 20-50 million cells, gently suspend the cells, and ice bath for 10-15 minutes. Transfer the cell suspension to a glass homogenizer, homogenize 6 times (10 seconds each time, stop for 10 seconds), and centrifuge the cell homogenate at 600g at 4℃ for 10 minutes. Carefully transfer the supernatant to a new centrifuge tube, centrifuge at 11000g at 4℃ for 10 minutes, and the precipitate is the cell mitochondria. Add protein lysis buffer containing 1% protease inhibitors and phosphatase inhibitors, place on ice for 10 minutes, and use a cell ultrasonic disruptor to fully lyse the mitochondria. Centrifuge the cells at 12000rpm at 4℃ for 15 minutes, aspirate the supernatant, and transfer to a new centrifuge tube. The protein concentration was determined using the Bio-Tech BCA protein detection kit, and the concentration of each sample was adjusted to the same using a protein lysis buffer containing protease inhibitors and phosphatase inhibitors. 1 / 4 of the sample volume of 5× protein loading buffer was added and placed in a -20°C refrigerator for later use. Protein expression was detected using Western blot.

[0114] The method for preparing the cell protein for Western blot detection in the above embodiment is as follows:

[0115] Discard the old cell culture medium and wash the cells twice with pre-cooled saline. Puff the cells with 1 mL of saline, centrifuge at 3000 rpm for 5 minutes, and discard the supernatant. Add protein lysis buffer containing 1% protease inhibitors and phosphatase inhibitors, place on ice for 10 minutes, and use a cell ultrasonic disruptor to fully lyse the cells. Centrifuge the cells at 12000 rpm and 4°C for 15 minutes, aspirate the supernatant, and transfer to a new centrifuge tube. Use the Biyuntian BCA protein detection kit to determine the protein concentration, and adjust the concentration of each sample to the same using a protein lysis buffer containing protease inhibitors and phosphatase inhibitors, add 1 / 4 of the sample volume of 5× protein loading buffer, and place in a -20°C refrigerator for use.

[0116] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of substances that supplement or upregulate OXA1L in (I) or (II): (I) Preparation of products for alleviating or treating autosomal dominant optic atrophy; (II) Prepare products to alleviate or treat mitochondrial dysfunction caused by AFG3L2 mutation.

2. The use according to claim 1, characterized in that: The autosomal dominant optic atrophy is caused by mutations in AFG3L2; Optionally, the AFG3L2 mutation comprises a mutation in the AFG3L2 intermembrane domain; Optionally, the AFG3L2 mutation includes AFG3L2 F175 mutation; Optionally, the AFG3L2 F175 Mutations include AFG3L2 F175S mutation.

3. The use according to claim 1, characterized in that: The mitochondrial dysfunction caused by the AFG3L2 mutation includes mitochondrial dysfunction caused by the AFG3L2 intermembrane domain mutation; Optionally, the AFG3L2 mutation includes AFG3L2 F175 mutation; Optionally, the AFG3L2 F175 Mutations include AFG3L2 F175S mutation.

4. The use according to claim 1, characterized in that: The alleviation or treatment of autosomal dominant optic atrophy and the alleviation or treatment of mitochondrial dysfunction caused by AFG3L2 mutation respectively and independently include one or more of reducing cellular ROS level, reducing mitochondrial ROS level and increasing mitochondrial membrane potential.

5. The use according to claim 1, characterized in that: The subject of the product includes a mammal or a cell derived from a mammal.

6. The use according to any one of claims 1 to 5, characterized in that: The substance that supplements or upregulates OXA1L includes at least one of OXA1L protein, a polynucleotide encoding the OXA1L gene, and an OXA1L agonist, wherein the OXA1L agonist promotes the expression of the OXA1L gene and / or protein synthesis in the subject; Optionally, the substance for supplementing or upregulating OXA1L comprises a vector carrying the gene encoding OXA1L.

7. The use according to any one of claims 1 to 5, characterized in that: The product also includes substances that supplement or upregulate SOD2.

8. The use according to claim 7, characterized in that: The substance for supplementing or upregulating SOD2 includes at least one of SOD2 protein, a polynucleotide encoding a SOD2 gene, and a SOD2 agonist, wherein the SOD2 agonist promotes SOD2 gene expression and / or protein synthesis in the subject; Optionally, the substance for supplementing or upregulating SOD2 includes a vector carrying the gene encoding SOD2.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition is used to alleviate or treat autosomal dominant optic atrophy; or to alleviate or treat mitochondrial dysfunction caused by AFG3L2 mutation; the pharmaceutical composition comprises a substance that supplements or upregulates OXA1L, and a substance that supplements or upregulates SOD2.

10. The pharmaceutical composition according to claim 9, characterized in that The substance that supplements or upregulates OXA1L includes at least one of OXA1L protein, a polynucleotide encoding the OXA1L gene, and an OXA1L agonist, wherein the OXA1L agonist promotes the expression of the OXA1L gene and / or protein synthesis in the subject; And / or, the substance that supplements or upregulates SOD2 includes at least one of SOD2 protein, a polynucleotide encoding a SOD2 gene, and a SOD2 agonist, and the SOD2 agonist promotes SOD2 gene expression and / or protein synthesis in the subject.

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