CN115770243B

CN115770243B - Application of compound DNJ in preparation of medicine for promoting OPA1 dimer formation

Info

Publication number: CN115770243B
Application number: CN202111036977.5A
Authority: CN
Inventors: 严庆丰; 庄倩倩; 孙亚萍; 胡爽依
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2021-09-06
Filing date: 2021-09-06
Publication date: 2024-03-26
Anticipated expiration: 2041-09-06
Also published as: CN115770243A

Abstract

The invention discloses an application of a compound DNJ in preparing a medicament for promoting OPA1 dimer formation and an application of the compound DNJ in preparing a medicament for treating diseases related to OPA1 dimer formation imbalance. According to the invention, the compound DNJ can promote the formation of OPA1 dimer by targeting OPA1, repair mitochondrial ultrastructure, obviously save mitochondrial function and effectively improve cell physiological state. The compound DNJ can be used for preparing medicines for promoting the formation of OPA1 dimer or medicines for treating diseases related to imbalance of OPA1 dimer formation, for example, DNJ can be used as potential therapeutic medicines for MT-RNR2 mutation related hypertrophic cardiomyopathy and MT-RNR1 mutation related deafness.

Description

Application of compound DNJ in preparation of medicine for promoting OPA1 dimer formation

Technical Field

The invention relates to the technical field of biological medicine, in particular to application of a compound DNJ in preparation of a medicament for promoting OPA1 dimer formation.

Background

Mitochondria are not only important sites of cellular energy metabolism, but also with apoptosis, reactive oxygen species production and Ca ²⁺ Steady state, etc. Mitochondrial function is co-regulated by nuclear and mitochondrial genes (mtDNA), most of which are encoded by nuclear genes, of about 1500 mitochondrial proteins, mtDNA encoding only 13 mitochondrial electron transport chain complex subunits, 22 trnas, and 2 rrnas. Mitochondrial dysfunction, particularly electron transport chain dysfunction, can exhibit a variety of clinical phenotypes, collectively referred to as mitochondrial disease, with a morbidity of approximately 1/6500. m.11778G was reported by Wallace et al 1988>The A mutation is an important causative factor for Lerber's hereditary optic neuropathy (Lerber's hereditary optic neuropathy, LHON), and hundreds of mtDNA mutations associated with mitochondrial disease have been discovered so far.

Hypertrophic cardiomyopathy (hypertrophic cardiomyopathy, HCM) is a primary cardiomyopathy characterized by asymmetric hypertrophy of the left ventricle and/or ventricular septum, and is one of the main causes of sudden cardiac death in teenagers and athletes worldwide, with a morbidity of about 0.05% -0.2%. Familial inheritance of HCM is mainly represented by autosomal dominant inheritance caused by mutations in the myocardial sarcomere protein gene. Some HCM cases have maternal genetic characteristics, associated with mtDNA mutations. A novel mutation of the HCM-related mitochondrial MT-RNR2 gene was previously identified in this laboratory (Liu Z, song Y, li D, et al, the novel mitochondrial 16S rRNA 2336T>C mutation is associated with hypertrophic cardiomyopathy,Journal of Medical Genetics,2014;51:176-184.) and was successful in constructing Induced Pluripotent Stem Cells (iPSCs) and their committed differentiated cardiomyocytes (iPSC-CMs) (Li S, pan H, tan C, et al, mitocondral dysfunctions contribute to hypertrophic cardiomyopathy in patient iPSC-derived cardiomyocytes with MT-RNR2 mutation, stem Cell reports.2018; 10:808-821.). Patient-specific iPSCs committed differentiated cardiomyocytes exhibited significant mitochondrial dysfunction and had characteristics similar to those associated with HCM cardiomyocytes.

Deafness is a major public health problem, and about 3.6 million deaf people worldwide account for about 5% of the world population. Deafness is mainly caused by genetic factors, environmental factors or both. mtDNA mutation is one of the important causes of deafness, wherein the MT-RNR1 gene m.1555A > G mutation is the main molecular basis for inducing non-syndrome type deafness by aminoglycoside antibiotics. iPSCs carrying the m.1555a > G mutation were constructed earlier in the laboratory and differentiated into neurons. Neurons carrying this mutation exhibit significant mitochondrial dysfunction.

Optic atrophy proteins (optic atrophy protein-1, opa1) are a class of dynamins, primarily localized to the inner mitochondrial membrane, and OPA1 multimers are critical for maintaining mitochondrial morphology. OPA1 dysfunction can lead to mitochondrial ridge disorders, mitochondrial division, etc., and further cause a series of diseases such as optic atrophy. At present, drugs targeting OPA1 are few and most of them are inhibitors. It has been reported that MYLS22 achieves the effect of inhibiting tumor growth by inhibiting OPA1 expression.

1-Deoxynojirimycin (DNJ) is a polyhydroxy alkaloid, is the main active ingredient of mulberry leaves, and has a certain blood sugar reducing effect. DNJ has a molecular structure similar to that of glucose and can compete for binding to alpha-glucosidase, thereby inhibiting postprandial elevation of blood glucose levels. In addition, DNJ can promote lipid metabolism and has a certain protection effect on liver lipid abnormality caused by high-fat diet.

Currently, DNJ completes phase II clinical trials in rare disease pompe disease (also known as acid alpha-glucosidase deficiency). The research shows that DNJ-combined rhGAA (human recombinant acid alpha-glucosidase) enzyme replacement therapy injection has good development prospect when being used for pompe patients. DNJ is used as a pharmacological molecular chaperone of rhGAA, and can play a role in stabilizing enzymes in blood and keeping the enzymes active. And combination therapy of AT2221, the hydrochloride form of DNJ, with rhGAA has been approved by the us FDA for breakthrough therapy in month 2 of 2019 for the treatment of late-onset pompe disease. DNJ also starts a brand-new angle of head on the stage of cardiovascular disease. 144 patients with blood stasis were enrolled by Ma et al (Ma Y, lv W, gu Y, et al 1-Deoxynojirimycin in Mulberry (Morus indica L.) Leaves Ameliorates Stable Angina Pectoris in Patients With Coronary Heart Disease by Improving Antioxidant and Anti-inpatient Capatients.front Pharmacol.2019 May 21; 10:569.) and were frequently suffering from angina pectoris. After 4 weeks of treatment with DNJ, the left ventricular ejection fraction of the experimental group was found to be significantly increased, the left ventricular mass index was significantly reduced, and both the aortic dilatability and the atherosclerosis index were significantly improved. Furthermore, intervention of DNJ can increase walking distance without angina, improve angina frequency, etc. Zhao et al (Zhao Q, jia TZ, cao QC, et al A Crude 1-DNJ Extract from Home Made Bombyx Batryticatus Inhibits Diabetic Cardiomyopathy-Associated Fibrosis in db/db Mice and Reduces Protein N-glycation levels, int J Mol Sci.2018 Jun 7;19 (6): 1699.) found that DNJ significantly down-regulates N-Glycosylation of diabetic mouse myocardial proteins, thereby alleviating the extent of Diabetic Cardiomyopathy (DCM) myocardial fibrosis.

The efficacy of DNJ in hypertrophic cardiomyopathy and deafness has not been reported at present, and drugs targeting agonists of OPA1 have not been reported.

Disclosure of Invention

The method is based on the research of pathogenic mechanisms of pre-MT-RNR 2 mutation related hypertrophic cardiomyopathy and MT-RNR1 mutation related deafness, takes cardiomyocytes differentiated from iPSCs of patients with the MT-RNR2 mutation and neurons differentiated from iPSCs of patients with the MT-RNR1 mutation as models, and can obviously save mitochondrial functions and effectively improve cell physiological states through treatment of a micromolecular compound DNJ. The drop-down experiment and the protein crosslinking experiment show that DNJ promotes the formation of OPA1 dimer by targeting OPA1, and repairs the mitochondrial ultrastructure. The application considers DNJ to be used as a potential therapeutic drug for MT-RNR2 mutation related hypertrophic cardiomyopathy and MT-RNR1 mutation related deafness.

The invention firstly provides application of a compound DNJ in preparing medicines for promoting OPA1 dimer formation.

The invention also provides application of the compound DNJ in preparing medicines for treating diseases related to imbalance of OPA1 dimer formation.

Preferably, the application mentioned, the disease associated with imbalance of OPA1 dimer formation is hypertrophic cardiomyopathy, deafness or optic atrophy. Further preferably, the hypertrophic cardiomyopathy is caused by mutation of MT-RNR2 gene. Further preferably, the deafness is caused by mutation of MT-RNR1 gene. Mutations in the MT-RNR2 gene or MT-RNR1 gene cause mitochondrial dysfunction.

Preferably, DNJ is used in an amount of 10 to 100. Mu. Mol/L.

The invention also provides a medicine for treating diseases related to imbalance of OPA1 dimer formation, and the active ingredient is a compound DNJ. Preferably, the disorder associated with imbalance in OPA1 dimer formation is hypertrophic cardiomyopathy, deafness or optic atrophy. More preferably, the hypertrophic cardiomyopathy is caused by mutation of the MT-RNR2 gene; the deafness is caused by mutation of MT-RNR1 gene.

According to the invention, the compound DNJ can promote the formation of OPA1 dimer by targeting OPA1, repair mitochondrial ultrastructure, obviously save mitochondrial function and effectively improve cell physiological state. The compound DNJ can be used for preparing medicines for promoting the formation of OPA1 dimer or medicines for treating diseases related to imbalance of OPA1 dimer formation, for example, DNJ can be used as potential therapeutic medicines for MT-RNR2 mutation related hypertrophic cardiomyopathy and MT-RNR1 mutation related deafness.

Drawings

FIG. 1 is a graph showing the results of immunofluorescence detection of cardiomyocytes differentiated from MT-RNR2 wild-type and mutant iPSCs.

FIG. 2 is a graph showing the results of detection of the effect of DNJ on mitochondrial oxygen consumption of MT-RNR2 mutant HCM-iPSC-CMs. A: indicating the oxygen consumption of the cells after the addition of different ETC targeted drugs; b: and (3) obtaining the basic oxygen consumption of the cell, the oxygen consumption of the coupled ATP, the maximum oxygen consumption and the residual oxygen consumption according to the graph A. n=9, P <0.01, P <0.001.

FIG. 3 is the effect of DNJ on mitochondrial activity of MT-RNR2 mutant HCM-iPSC-CMs. A: the viability of the cells on different days is shown; b: the cell viability on the third day of dosing was shown. n=3, P <0.01.

FIG. 4 shows the effect of DNJ on recovery of MT-RNR2 mutant HCM-iPSC-CMs action potential. A: a cardiomyocyte electrophysiological representation representing different treatments; b: and counting the electrophysiological conditions of the myocardial cells in different time courses. Wild type + DMSO, n=12; mutant + DMSO, n=14; mutant +dnj, n=14. * P <0.05, P <0.01.

FIG. 5 is a DNJ coupled carboxyl magnetic bead in vitro pulldown experiment.

FIG. 6 shows the effect of DNJ on OPA1 multimers of MT-RNR2 mutant HCM-iPSC-CMs.

FIG. 7 is the effect of DNJ on mitochondrial ridge morphology of MT-RNR2 mutant HCM-iPSC-CMs.

FIG. 8 is a graph showing the results of immunofluorescence detection of neurons differentiated from MT-RNR1 wild-type and mutant iPSCs.

FIG. 9 shows the effect of DNJ on restoring action potential of MT-RNR1 mutant neurons. A: representing the behavior of neurons under different treatments; b: counting action potential amplitude, delay time, action potential rising time and action potential time course. Wild type + DMSO, n=16; mutant + DMSO, n=19; mutant +dnj, n=8. * P <0.05, P <0.01.

Detailed Description

Example 1

Urine cells from patients with hypertrophic cardiomyopathy harboring MT-RNR2 mutations are induced to iPSCs and differentiated to iPSC-CMs.

Establishment and identification of HCM patient-specific iPSCs (HCM-iPSCs) carrying MT-RNR2 mutations. Urine cells from stock members of the HCM family and normal control individuals were collected and infected with retrovirus to establish iPSCs (see earlier published papers by the inventors: li S, pan H, tan C, et al Mitocondral dysfunctions contribute to hypertrophic cardiomyopathy in patient iPSC-derived cardiomyocytes with MT-RNR2 mutant Cell reports.2018; 10:808-821.).

Directed differentiation of HCM patient-specific cardiomyocytes (HCM-iPSC-CMs) carrying MT-RNR2 mutations. iPSC in vitro myocardial directional differentiation was performed using a 2D monolayer differentiation technique based on small molecule compounds. 3-4 days before induced differentiation, iPSCs were digested into single cells with Accutase (Stem cell) and resuspended in mTESR1 (Stem cell) culture medium, 10 ⁵ Individual cells were plated uniformly on Matrigel (BD) plated six well plates. On day 0, when the cell density reached about 95%, RPMI/B27-insulin (Gibco, cat. No. A1895601) +12. Mu.M CHIR99021 (selleck, cat. No. C.T 99021) was changed and cultured for 24 hours. On day 1, CHIR99021 was removed from the cells and the culture was continued by changing to RPMI/B27-insulin medium. On days 2-3, 5. Mu. Mol/L IWP2 (Tocris, cat. No. 3533) was given to the cells in RPMI/B27-insulin for 2 days. After the IWP2 and RPMI/B27-insulin culture solution in the cells are removed from the cells for 2 days on days 3-4, the culture solution is changed into RPMI/B27 (Gibco) for 7-12 days, and spontaneously beating myocardial cells can be observed successively.

The 24 wells were plated with a slide and the cells were seeded. After three washes with PBS, 4% paraformaldehyde was added and the mixture was fixed at room temperature for 15min. Then 0.2% Triton-X100 was added and the mixture was allowed to pass through at room temperature for 15min. Then 3% BSA was added and the mixture was blocked at room temperature for 1 hour. MLC2v (Abcam) was added overnight at 4 ℃. Adding fluorescent secondary antibody (the secondary antibody is Goat Anti-Rabbit IgG H)&L(Alexa488 (Abcam). ) Incubate for 1h at room temperature in the dark. Further, 0.5mL of 1. Mu.g/mL DAPI was added, left at room temperature in the dark for 5min, and blocked with 50% glycerol. During this period each step was washed 3 times with PBS. Finally, the results were observed under a confocal microscope. The results are shown in FIG. 1, and the immunofluorescence results show that the induced cardiomyocytes can successfully express the cardiac marker protein MLC2v, indicating that the above steps can successfully differentiate HCM-iPSCs carrying MT-RNR2 mutation into cardiomyocytes.

Example 2

DNJ the HCM-iPSC-CMs carrying MT-RNR2 mutations obtained in example 1 were treated and their mitochondrial oxygen consumption was determined.

Cells were seeded at 5000 cells/well in 96-well plates and placed at 37℃in 5% CO ₂ An incubator. After 16h, the experimental group cells were replaced with 30. Mu. Mol/L DNJ (Targetmol) in RPMI/B27 medium and the control group cells were replaced with equal volumes of DMSO. Oligomycin, FCCP, rotenone/antimycin A was added sequentially. Oligomycin (Seahorse XF): the drug inhibits ATP synthase (i.e., mitochondrial complex v), and the first addition after measurement of cellular basal respiration can affect or reduce electron flow through ETC, causing mitochondrial respiration or OCR reduction, which in turn is associated with cellular ATP synthesis. FCCP: the medicine is added after oligomycin and is a decoupling agent, and the adding of the medicine can destroy proton gradient and mitochondrial membrane potential, so that electrons are transmitted in an electron transmission chain without limitation, and meanwhile, the oxygen consumption of the compound IV reaches the maximum. FCCP-stimulated OCR can be used to calculate the cell's reserve breath capacity (which is the difference between the maximum breath and the basal breath), which represents the ability of the cell to respond to an increase in energy demand or under pressure. rotenone/antimycin a: the third drug added was a mixture of rotenone and antimycin A. Rotenone is an inhibitor of complex i and antimycin a is an inhibitor of complex iii. These two drugs shut down mitochondrial respiration, allowing the calculation of non-mitochondrial respiratory oxygen consumption driven by extra-mitochondrial activity. Basic oxygen consumption:for satisfying ATP requirements of cells and oxygen consumption of proton leak. Representing the energy demand of the cell in the basal state. Coupled ATP oxygen consumption: the oxygen consumption reduction part generated after the addition of oligomycin accounts for a part of the oxygen consumption of the basic respiration and is used for driving ATP synthesis. Representing the ATP synthesis capacity of mitochondria to meet cellular energy demands. Maximum oxygen consumption: maximum oxygen consumption of cells obtained after FCCP addition. FCCP mimics a physiological "energy requirement" by stimulating the cellular respiratory chain to work at maximum capacity, which causes rapid oxidation of substrates (sugars, fats, amino acids) to address this metabolic challenge. Representing the maximum respiration rate that the cell can achieve. Residual oxygen consumption: the maximum breath minus the oxygen consumption of the basal breath. The ability of a cell to respond to demand can be an indicator of cell fitness or flexibility, representing the potential response of the cell to energy demand and the gap between the basal respiration of the cell and the theoretical respiration maximum.

The results show that the basic oxygen consumption, the maximum oxygen consumption and the residual oxygen consumption of the HCM-iPSC-CMs are all obviously lower than those of the wild iPSC-CMs; the above indicators of HCM-iPSC-CMs were all significantly increased by DNJ treatment (FIG. 2A; 2B). It was shown that DNJ treatment restored mitochondrial function of HCM-iPSC-CMs carrying MT-RNR2 mutations.

Example 3

DNJ treatment the HCM-iPSC-CMs carrying MT-RNR2 mutations obtained in example 1 were tested for their effect on cell viability by galactose-induced cell death experiments.

The cells were grown in 2X 10 cells ⁴ Inoculating the cells/well to 24-well plate, culturing with L-15 medium (Gibco) and B27 cell culture additive, standing at 37deg.C, 5% CO ₂ An incubator. After 16h, the experimental group cells were replaced with L-15 medium supplemented with drug (final concentration 30. Mu. Mol/L), the control group cells were replaced with an equal volume of DMSO, and 3 duplicate wells were set per treatment. Cells from each well were collected every 24 hours, counted with a cytometer, and counted 3 times in succession. Glycogen in L-15 medium is mainly galactose, and in this culture environment, cells are mainly supplied with energy through mitochondria.

The results showed that the survival of HCM-iPSC-CMs carrying MT-RNR2 mutation was lower in L-15 medium compared to wild type and significantly improved upon DNJ addition (FIG. 3A; 3B). DNJ was shown to enhance cell viability by improving mitochondrial function.

Example 4

DNJ the HCM-iPSC-CMs carrying MT-RNR2 mutations obtained in example 1 were treated and tested for electrophysiology.

Cell slide was plated on 24-well plates and incubated with Matrigel (BD) for 1h. Cardiomyocytes were digested with TrypLE (Gibco), incubated at 37 ℃, and the supernatant was discarded by centrifugation. The cells were spread on 24-well plate climbs at 37℃with 5% CO ₂ After 16 hours of incubation, the solution was changed, the final concentration of drug was 30. Mu. Mol/L in the experimental group and the equivalent volume of DMSO in the control group. Culturing was continued for 48 hours.

Taking out the cell climbing sheet, placing the cell climbing sheet in an inverted microscope groove with a constant temperature perfusion groove at 37 ℃, and continuously perfusing with extracellular fluid. Spontaneous action potentials of cardiomyocytes were recorded using whole-cell patch clamp technique. The patch clamp amplifier employs a 700B amplifier. The glass microelectrode is filled with electrode inner liquid, the electrode resistance used when recording action potential is 3-5MΩ, and high-resistance (-5 GΩ) seal is formed between the electrode and cell membrane. The cell membrane is sucked and broken by negative pressure, capacitance and series resistance compensation are regulated, the sampling frequency is l0k Hz, the low-pass filtering frequency is 2k Hz, and the action potential of the spontaneously beating myocardial cells is recorded after the electrode inner liquid and the cell inner liquid are balanced for 5min. Data were analyzed using pClamP 10.2 and Lab Chart 8.0 software.

The results show that the action potential time course (APDs) of HCM-iPSC-CMs carrying MT-RNR2 mutation is significantly longer than that of wild type; APDs were significantly shortened by DNJ treatment (fig. 4a;4 b). DNJ was shown to improve the electrophysiological status of mutated cardiomyocytes.

Example 5

And (3) performing an in-vitro pull-down experiment of the cell lysate by coupling DNJ to the magnetic beads, and verifying the drug target.

Preparation of DNJ-coupled carboxyl magnetic beads: experimental procedures refer to the product specifications of the Promega company. That is, carboxyl magnetic beads were reacted with imino groups of DNJ in MES buffer solution (ph=6.0) containing EDC catalyst, reacted overnight at room temperature, and the reaction was terminated after PBST washing and stored at 4 ℃.

Exogenous Pull down experiment: after 293T over-expressing EGFP and OPA1 was collected, cleavage was performed, and after supernatant was incubated with beads and DNJ-beads for 4h at 4℃the supernatant was washed three times with lysis buffer, and finally mixed with 40. Mu.L SDS loading buffer and boiled for 10min, and Western blot analysis of binding between EGFP and OPA1 and DNJ-beads was performed.

The results are shown in FIG. 5, where DNJ was able to bind over-expressed OPA1 in vitro, whereas negative control EGFP was unable. Indicating that there is an interaction between DNJ and OPA 1.

Example 6

DNJ the MT-RNR2 mutation-carrying HCM-iPSC-CMs obtained in example 1 were treated and tested for multimeric status on OPA 1.

Mitochondria are extracted from the induced myocardial cells, 100 mu L of KPBS containing 10mmol/L EDC is added for resuspension, the mixture is kept stand for 30min at room temperature, 15mmol/L DTT is added for stopping the reaction, and the mitochondria are lysed for protein extraction. Finally obtaining a result through western blotting.

As shown in fig. 6, DNJ treatment significantly increased the multimeric formation of the mutated cardiomyocyte OPA 1. It was shown that DNJ functions by targeting OPA1, promoting OPA1 multimer formation.

Example 7

DNJ the MT-RNR2 mutation-carrying HCM-iPSC-CMs obtained in example 1 were treated and the morphology of the mitochondrial ridge was observed.

Cells were washed once with PBS and collected with a cell scraper. 1mL of DPBS was added for resuspension, 3000rpm, and centrifugation was performed for 5min. The supernatant was discarded, 0.5mL of 2.5% glutaraldehyde was added, and the cells were transferred to a 1.5mL centrifuge tube and fixed for 1h.3000rpm, and centrifuged for 5min. Glutaraldehyde was removed as much as possible, rinsed 2 times with 500. Mu.L of 0.1M PBS, and placed at 4℃for 10-15min each time. PBS was removed, cells were blown off by adding 1% osmium acid, and the cells were fixed at 4℃for 1 hour. 3000rpm, and centrifuged for 5min. Add 500. Mu.L ddH ₂ O was rinsed 2 times for 15min each. Dyeing with 2% uranium acetate aqueous solution, and standing at 4deg.C for 30min. Gradient dehydration: sequentially adding 50%, 70% and 90% ethanol for dehydration, and standing at 4deg.C for 10min. And dehydrating with 100% alcohol for 20min.100% acetone was dehydrated 2 times for 20 min/time. Mixing and infiltrating anhydrous acetone and embedding agent according to the volume of 1:1The sample is permeated and placed at normal temperature for 2 hours. Embedding with pure embedding agent, and polymerizing in an oven. 37 ℃ for 24 hours; 45 ℃ for 24 hours; 68 ℃ for 48h. Polymerization in an oven: 37 ℃ for 24 hours; 45 ℃ for 24 hours; 68 ℃ for 48h. Slice staining: slicing by an ultrathin slicing machine, dyeing by 4% uranium acetate for 20min and dyeing by lead citrate for 5min. And (5) carrying out electron microscope observation on the sample.

The results are shown in FIG. 7, where DNJ treatment encouraged more ordered mitochondrial ultrastructural organization of HCM-iPSC-CMs, with richer and tighter mitochondrial spines.

Example 8

Urine cells of deaf patients carrying MT-RNR1 mutation were induced into iPSCs, and iPSCs were differentiated into neurons by a two-step differentiation method of iPSCs-neural stem cells-auditory neurons.

The day before induced differentiation of neural Stem cells, iPSCs were digested into single cells with Accutase (Stem cell) and resuspended in mTESR1 (Stem cell) culture medium, 2.5-3.0X10 ⁵ Individual cells were plated uniformly on Matrigel (BD) plated six well plates. On day 0, when the cell density reaches 10-20%, the culture medium is changed into a neural stem induction culture medium (Gibico, cat. No. A1647801) to be continuously induced for 6 days, and liquid is changed every two days. The neural stem cells of the P0 generation are obtained on the 7 th day, and the cells are passaged. After digesting the neural stem cells with Ackutase, the neural stem cells were resuspended in neural stem cell expansion medium, and 1.5X10 ⁶ Cells were seeded on six well plates with Geltrex (Gibco, cat. No. A1413302) plated, and thereafter cultured with neural stem cell expansion medium at all times, with fluid changes every two days. And when the confluence reaches 80% -90%, the neural stem cells can be subjected to passage and cryopreservation. Neural stem cell induction media included neural basal media and neural induction supplements (50×). Neural stem cell expansion medium includes neural basal medium: DMEM/F12 (Gibco, cat#11330-057) =1:1 and neuro-induction supplement (50×).

The day before the induction of the differentiation of neurons, the neural stem cells were digested with Actutase and resuspended in neural stem cell culture medium, and 1X 10 ⁵ Cells were seeded on six well plates with a bed of laminin (10. Mu.g/mL) (Gibco, cat. No. 23017-015). On day 0, an auditory neuron differentiation medium was added, followed by a fluid change every other day. Neuron differentiation medium includes neural basal medium (Gibico, cat. No. 21103049)B-27 (50×) (Gibico, cat. No. A3582801), glutamine (Glutamax) (100×) (Gibico, cat. No. 35050061), nonessential amino acids (NEAA) (100×) (Gibico, cat. No. 11140050), brain-derived neurotrophic factor (BDNF) (R) 20ng/mL&D, cat.no. 248-BDB), 20ng/ml glial cell line-derived neurotrophic factor (GDNF) (R&D, cat.no. 212-GD) and 200 μg/mL L-Ascorbic Acid (AA) (Sigma-Aldrich, cat.no. 50-81-7). After 14 days of culture, experiments can be performed.

The 24 wells were plated with a slide and the cells were seeded. After three washes with PBS, 4% paraformaldehyde was added and the mixture was fixed at room temperature for 15min. Then 0.2% Triton-X100 was added and the mixture was allowed to pass through at room temperature for 15min. Then 3% BSA was added and the mixture was blocked at room temperature for 1 hour. TrkB (Abcam, cat#ab 18987), TUJ1 (Abcam, cat#ab 7751), neuN (Abcam, cat#ab 177487) were added overnight at 4 ℃. Adding fluorescent secondary antibody (the secondary antibody is Goat Anti-Rabbit IgG H)&L(Alexa488 (Abcam)), incubated at room temperature for 1h in the dark. Further, 0.5mL of 1. Mu.g/mL DAPI was added, left at room temperature in the dark for 5min, and blocked with 50% glycerol. During this period each step was washed 3 times with PBS. Finally, the results were observed under a confocal microscope. The results are shown in fig. 8, and the immunofluorescence results show that the induced neurons can successfully express the neuron marker protein TrkB, neuN, TUJ1. The above procedure was shown to successfully differentiate iPSCs carrying MT-RNR1 mutation into neurons.

Example 9

DNJ neurons carrying MT-RNR1 mutations obtained in example 8 were treated and tested for electrophysiology.

The cell climbing sheet is paved on a 24-well plate, and is incubated for 1h by adding laminin. Neuronal cells were digested with Ackutase and the supernatant was discarded by centrifugation. The cells were spread on 24-well plate climbs at 37℃with 5% CO ₂ After 16 hours of incubation, the solution was changed, the drug was added to the experimental group at a final concentration of 30. Mu.g/mL, and the control group was added with an equal volume of DMSO. Culturing was continued for 48H. The cell climbing sheet is taken out from the culture dish and placed in an inverted microscope trough with a constant temperature perfusion trough at 37 ℃ to carry out continuous perfusion by extracellular fluid. Using an electrode drawing instrumentThe glass microelectrode is pulled to an opening of about 1 mu M, about 1/3 volume of electrode internal liquid is poured, the microelectrode is installed to a recording probe, and the liquid inlet resistance is 6-8MΩ after the electrode liquid is poured. After applying positive pressure of 10mmHg to the microelectrode, the micromanipulation was adjusted to bring it close to the cell. When the electrode is close to the cell and the electrode resistance is increased, the positive pressure is removed, the cell clamping voltage is adjusted to minus 10mV, meanwhile, a small negative pressure is added to the cell, and the negative pressure is removed after the high-resistance sealing is formed. After the sealing is stable, the cells are clamped at-70 mV, a large negative pressure is rapidly given, and meanwhile, an electric shock rupture membrane is added, so that a whole cell sealing state is formed, and the cells with the membrane potential near-65 mV are selected and recorded in a current clamp mode. In the current clamp mode, 0-90pA of current is infused into the cells, the current amplification is 10pA, and the response of the cells to depolarization current is recorded.

The results showed that neurons harboring MT-RNR1 mutations were treated with DNJ for 48h, and that the action potential depolarization current threshold decreased, and the action potential morphology was improved under 90pA current stimulation, as evidenced by an increase in action potential amplitude, a decrease in rise time, and a decrease in time course (FIG. 9A; 9B). DNJ was shown to significantly improve the function of mutant auditory neurons.

Claims

1. The application of the compound DNJ in preparing medicaments for treating diseases related to unbalanced OPA1 dimer formation,

the diseases related to imbalance of OPA1 dimer formation are hypertrophic cardiomyopathy, deafness or optic atrophy.

2. The use according to claim 1, wherein the hypertrophic cardiomyopathy is caused by MT-RNR2 gene mutation.

3. The use according to claim 1, wherein the deafness is caused by mutation of MT-RNR1 gene.

4. The use according to claim 2 or 3, wherein mutation of the MT-RNR2 gene or the MT-RNR1 gene causes mitochondrial dysfunction.

5. The use according to claim 1, wherein DNJ is used in an amount of 10 to 100. Mu. Mol/L.