CN115770243A

CN115770243A - Application of compound DNJ in preparation of medicine for promoting formation of OPA1 dimer

Info

Publication number: CN115770243A
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: 2023-03-10
Anticipated expiration: 2041-09-06
Also published as: CN115770243B

Abstract

The invention discloses application of a compound DNJ in preparing a medicament for promoting formation of OPA1 dimer and application of the compound DNJ in preparing a medicament for treating diseases related to imbalance of formation of OPA1 dimer. The research of the invention discovers that the compound DNJ can promote the formation of OPA1 dimer, repair mitochondrial ultrastructure, remarkably save mitochondrial function and effectively improve cell physiological state by targeting OPA 1. The compound DNJ can be used for preparing a medicine for promoting the formation of OPA1 dimer or preparing a medicine for treating diseases related to the imbalance of the formation of OPA1 dimer, for example, the DNJ can be used as a potential treatment medicine for MT-RNR2 mutation-related hypertrophic cardiomyopathy and MT-RNR1 mutation-related deafness.

Description

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

Technical Field

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

Background

Mitochondria are not only important sites of cellular energy metabolism, but also are involved in apoptosis, active oxygen production and Ca ²⁺ Steady state, etc. Mitochondrial function is regulated by both nuclear and mitochondrial genes (mtdnas), with the vast majority of the about 1500 mitochondrial proteins being encoded by nuclear genes, mtdnas encoding only 13 mitochondrial electron transport chain complex subunits, 22 trnas, and 2 rrnas. Mitochondrial dysfunction, and in particular electron transport chain dysfunction, presents with a variety of clinical phenotypes, collectively referred to as mitochondrial disease, with an incidence of about 1/6500. m.11778G was reported by Wallace et al in 1988>A mutation is an important causative factor of Lerber's Hereditary Optic Neuropathy (LHON), and hundreds of mtDNA mutations have been found to date to be associated with mitochondrial diseases.

Hypertrophic Cardiomyopathy (HCM) is a primary cardiomyopathy which is basically characterized by asymmetric hypertrophy of left ventricle and/or ventricular interval, is one of the main causes of sudden cardiac death of teenagers and athletes all over the world, and has the incidence rate of about 0.05-0.2%. HCM with familial inheritance is mainly characterized by autosomal dominant inheritance caused by mutation of myocardial sarcomeric protein genes. Some HCM cases have maternal genetic characteristics, associated with mtDNA mutations. A new HCM-related mitochondrial MT-RNR2 gene mutation (Liu Z, song Y, li D, et al. The novel mitochondrial 1696 rRNA 2336T > C mutation associated with pluripotent cardiac pathology, journal of Medical genetics,2014 51. The patient-specific iPSCs directionally differentiated cardiomyocytes showed significant mitochondrial dysfunction and had relevant characteristics similar to HCM cardiomyocytes.

Deafness is a major public health problem, and about 3.6 hundred million deaf people exist in the world, accounting for about 5 percent of the world population. Deafness is mainly caused by genetic factors, environmental factors or the combination of both. mtDNA mutation is one of important reasons for deafness, wherein m.1555A > G mutation of MT-RNR1 gene is the main molecular basis for causing aminoglycoside antibiotic-induced non-syndromic deafness. iPSCs carrying m.1555A > G mutation are constructed in the early stage of the laboratory and are differentiated into neurons. Neurons carrying this mutation show significant mitochondrial dysfunction and dysfunction.

Optic atrophy protein (OPA 1) is a group of dynamin, mainly localized in the inner mitochondrial membrane, and OPA1 multimer is critical for maintaining mitochondrial morphology. The functional defect of OPA1 can cause mitochondrial spine disorder, mitochondrial division and the like, and further cause a series of diseases such as optic atrophy and the like. At present, the OPA1 targeted drugs are few and most inhibitors. MYLS22 reportedly achieves tumor growth inhibition by inhibiting OPA1 expression.

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

Currently, DNJ has completed phase II clinical trials in rare disease pompe disease (also known as acid alpha-glucosidase deficiency). Research shows that the DNJ combined rhGAA (human recombinant acid alpha-glucosidase) enzyme replacement therapy injection has good development prospect when being used for patients suffering from pompe diseases. DNJ, as a pharmacological chaperone for rhGAA, can act to stabilize enzymes in blood and keep them active. And AT2221, the hydrochloride form of DNJ, in combination therapy with rhGAA has been approved by FDA awarding breakthrough therapy in the united states for the treatment of late-onset pompe disease in 2 months 2019. DNJ also began as a completely new horn in the arena of cardiovascular disease. Ma et al (Ma Y, lv W, gu Y, et al.1-Deoxynojirimycin in Mulberry (Morus indica L.) Leaves enzymes Stable antibiotic Pectonis in Patients With Coronary Heart Disease by Improving the inhibition and Anti-inflammatory disorders front Pharmacol.2019 May 21) recruited 144 Patients With blood stasis, who often had Angina Pectoris. After 4 weeks of DNJ treatment, the left ventricular ejection fraction of the experimental group is obviously increased, the left ventricular mass index is obviously reduced, and the aortic distensibility and the atherosclerosis index are obviously improved. In addition, intervention of DNJ can increase walking distance without angina, improve angina attack frequency, and the like. ZHao et al (ZHao Q, jia TZ, cao QC, et al. A CRude 1-DNJ Extract from Home Made Bombyx Batryticatus inhitis Inhibits Diabetic cardiac dynamics-Associated Fibrosis in db/db Rice and production Protein N-Glycosylation levels. Int J Mol Sci.2018 Jun 7 (6): 1699.) found that DNJ significantly down-regulated the N-Glycosylation of Diabetic mouse cardiac proteins, thereby alleviating the degree of Diabetic Cardiomyopathy (DCM) myocardial Fibrosis.

At present, the efficacy of DNJ in hypertrophic cardiomyopathy and deafness is not reported in research, and a medicament of an agonist targeting OPA1 is not reported.

Disclosure of Invention

Based on the pathogenic mechanism research of the hypertrophic cardiomyopathy related to the MT-RNR2 mutation and the deafness related to the MT-RNR1 mutation in the earlier stage, the iPSCs differentiated cardiac muscle cells of the hypertrophic cardiomyopathy patients carrying the MT-RNR2 mutation and the iPSCs differentiated neurons of the deafness patients carrying the MT-RNR1 mutation are used as models, and the models are treated by a small molecular compound DNJ, so that the mitochondrial function can be remarkably saved, and the cell physiological state can be effectively improved. Through a pull-down experiment and a protein crosslinking experiment, DNJ is found to promote the formation of OPA1 dimer and repair a mitochondrial ultrastructure by targeting OPA 1. The application considers that DNJ can be used as a potential therapeutic drug for MT-RNR2 mutation-related hypertrophic cardiomyopathy and MT-RNR1 mutation-related deafness.

The invention firstly provides the application of the compound DNJ in the preparation of the medicine for promoting the formation of OPA1 dimer.

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

Preferably, the use, the disease associated with an imbalance in OPA1 dimer formation is hypertrophic cardiomyopathy, deafness or optic atrophy. Further preferably, the hypertrophic cardiomyopathy is caused by a mutation in the MT-RNR2 gene. Further preferably, the deafness is caused by mutation of MT-RNR1 gene. Mitochondrial dysfunction is caused by MT-RNR2 gene or MT-RNR1 gene mutation.

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

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

The research of the invention discovers that the compound DNJ can promote the formation of OPA1 dimer, repair mitochondrial ultrastructure, remarkably save mitochondrial function and effectively improve cell physiological state by targeting OPA 1. The compound DNJ can be used for preparing a medicine for promoting the formation of OPA1 dimer or preparing a medicine for treating diseases related to the imbalance of the formation of OPA1 dimer, for example, DNJ can be used as a potential treatment medicine for MT-RNR2 mutation-related hypertrophic cardiomyopathy and MT-RNR1 mutation-related deafness.

Drawings

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

FIG. 2 is a diagram showing the result of detecting the effect of DNJ on the mitochondrial oxygen consumption of MT-RNR2 mutant HCM-iPSC-CMs. A: represents the situation of oxygen consumption of cells after the addition of different ETC targeting drugs; b: and (4) obtaining the basic oxygen consumption of the cells, 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 a graph showing the effect of DNJ on mitochondrial activity of MT-RNR2 mutant HCM-iPSC-CMs. A: indicates the viability of the cells at different days; b: indicating the cell viability on the third day of dosing. n =3, <0.01.

FIG. 4 shows the effect of DNJ on the action potential of MT-RNR2 mutant HCM-iPSC-CMs. A: electrophysiology representative maps of cardiomyocytes representing different treatments; b: and (5) 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 shows a DNJ-coupled in vitro pull-down assay with carboxyl magnetic beads.

FIG. 6 is a graph showing the effect of DNJ on OPA1 multimers of MT-RNR2 mutant HCM-iPSC-CMs.

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

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

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

Detailed Description

Example 1

The urine cells of patients with hypertrophic cardiomyopathy carrying MT-RNR2 mutation are induced into iPSCs and are differentiated into iPSC-CMs.

Establishment and identification of HCM patient-specific iPSCs (HCM-iPSCs) carrying MT-RNR2 mutation. Urine cells of HCM family maternal members and normal control individuals are collected and infected by retrovirus to establish iPSCs (the specific method is shown in a paper published by the inventor in the previous period: li S, pan H, tan C, et al. Mitochon mental dynamics functions control to hyperbaric cardiac pathology in patient iPSC-derived cardiac pathology with MT-RNR2 mutation. Stem Cell reports.2018; 10.

Directed differentiation of HCM patient-specific cardiomyocytes (HCM-iPSC-CMs) carrying MT-RNR2 mutations. Carrying out iPSC in-vitro myocardial directional differentiation by using a 2D monolayer differentiation technology based on a small molecular compound. 3-4 days before induced differentiation, iPSCs were digested into single cells with Accutase (Stem cell), resuspended in mTeSR1 (Stem cell) culture medium, and cultured for 10 days ⁵ Individual cells were plated evenly onto Matrigel (BD) plated six-well plates. On day 0, the cell density reachedAbout 95%, RPMI/B27-insulin (Gibco, cat. No. A1895601) +12 μ M CHIR99021 (seleck, cat. No. CT99021) was cultured for 24h. On day 1, CHIR99021 was removed from the cells and the cells were replaced with RPMI/B27-insulin medium for further culture. On days 2-3, cells were treated with 5. Mu. Mol/L IWP2 (Tocris, cat. No. 3533) in RPMI/B27-insulin for 2 days. On days 3-4, IWP2 was removed from the cells, and after 2 days of further culture in RPMI/B27-insulin medium, the medium was changed to RPMI/B27 (Gibco) on days 7-12, and spontaneously beating cardiomyocytes were observed successively.

The cells were seeded by plating in 24 wells. After washing three times with PBS, 4% paraformaldehyde was added and fixed at room temperature for 15min. Adding 0.2% Triton-X100, and allowing to permeate at room temperature for 15min. Then 3% BSA was added and blocked at room temperature for 1h. MLC2v (Abcam) was added and left overnight at 4 ℃. Then adding a fluorescent secondary antibody (the secondary antibody is Goat Anti-Rabbit IgG H)&L(Alexa

488 (Abcam). ) And incubating for 1h at room temperature in a dark place. Further, 0.5mL of 1. Mu.g/mL of DAPI was added, and the mixture was left at room temperature in the dark for 5min and mounted with 50% glycerol. During which each step was washed 3 times with PBS. And finally observing the result under a confocal microscope. The results are shown in figure 1, and the immunofluorescence results show that the induced cardiomyocytes can successfully express the cardiac marker protein MLC2v, which indicates that the steps can successfully differentiate HCM-iPSCs carrying MT-RNR2 mutation into cardiomyocytes.

Example 2

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

Inoculating cells at 5000/well into 96-well plates, standing at 37 ℃,5% CO ₂ An incubator. After 16h, the experimental cells were replaced with RPMI/B27 medium supplemented with 30. Mu. Mol/L DNJ (Targetmol) and the control cells were replaced with an equal volume of DMSO medium. Oligomycin, FCCP, rotenone/antimycin A were added in sequence. Oligomycin (Seahorse XF): the drug inhibits ATP synthase (i.e., mitochondrial complex V), and is added first after measuring basal respiration of cells, and the drug may influence or reduce electron flow through ETC, leadingThere is a reduction in mitochondrial respiration or OCR, which is associated with cellular ATP synthesis. FCCP: the drug is added after oligomycin, is an uncoupler, and can destroy proton gradient and mitochondrial membrane potential to cause the unrestricted transfer of electrons in an electron transfer chain, and simultaneously, the oxygen consumption of the compound IV reaches the maximum. FCCP stimulated OCR can be used to calculate the cell's reserve respiratory capacity (which is the difference between the maximum and basal breath) which represents the cell's ability to respond to an increase in energy demand or under pressure. rotinone/antimycin a: the third drug addition 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 can close mitochondrial respiration, thus enabling the calculation of non-mitochondrial respiration oxygen consumption driven by extra-mitochondrial activity. Basic oxygen consumption: used to meet the ATP requirements of the cells and the oxygen consumption of proton leakage. Representing the energy requirement of the cell in the basal state. Coupled ATP oxygen consumption: the oxygen consumption decline produced upon addition of oligomycin, which is a fraction of basal respiratory oxygen consumption, is used to drive ATP synthesis. Represents the ATP synthesis capacity of mitochondria to meet the energy requirements of the cell. Maximum oxygen consumption: the maximum oxygen consumption of the cells obtained after FCCP addition. FCCP mimics a physiological "energy demand" by stimulating the cellular respiratory chain to work at its 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. And (3) residual oxygen consumption: the maximum breath minus the oxygen consumption of the basal breath. The potential response capacity of the cell to the energy demand and the difference between the basic respiration of the cell and the theoretical respiration maximum are represented, and the capacity of the cell to respond to the demand can be used as an index of the adaptability or flexibility of the cell.

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 wild type iPSC-CMs; after DNJ treatment, the indexes of the HCM-iPSC-CMs are all obviously improved (FIG. 2A. It was shown that DNJ treatment could restore mitochondrial function to HCM-iPSC-CMs carrying the MT-RNR2 mutation.

Example 3

The HCM-iPSC-CMs carrying the MT-RNR2 mutation obtained in example 1 were treated with DNJ, and the effect of DNJ on cell viability was examined by a galactose-induced cell death experiment.

Cells were plated at 2X 10 ⁴ One/well inoculated in 24-well plates, incubated with L-15 medium (Gibco) and B27 cell culture additives, incubated at 37 ℃ and 5% CO ₂ An incubator. After 16h, the experimental group was changed to L-15 medium with drug (final concentration 30. Mu. Mol/L) and the control group was changed to equal volume of DMSO, with 3 duplicate wells per treatment. Cells from each well were collected every 24h, counted using a hemocytometer plate, and counted 3 times in succession. Glycogen in L-15 medium is mainly galactose, which in this culture environment would result in cells being supplied mainly by mitochondria.

The results show that the survival rate of HCM-iPSC-CMs carrying MT-RNR2 mutation in L-15 medium is lower than that of wild type, and is obviously improved after DNJ is added (FIG. 3A. DNJ was shown to increase cell viability by improving mitochondrial function.

Example 4

The HCM-iPSC-CMs carrying the MT-RNR2 mutation obtained in example 1 were treated with DNJ and subjected to electrophysiological detection.

Cell crawls were plated in 24-well plates and incubated for 1h with Matrigel (BD). The cardiomyocytes were digested with TrypLE (Gibco), incubated at 37 ℃ and centrifuged to discard the supernatant. Spreading the cells on a 24-well plate, 37 ℃,5% CO ₂ After 16 hours of incubation, the solution was changed, and the experimental group was added with a final concentration of 30. Mu. Mol/L drug, and the control group was added with an equal volume of DMSO. The culture was continued for 48h.

Taking out the cell slide, placing in an inverted microscope tank with a constant-temperature perfusion tank at 37 ℃, and continuously perfusing with extracellular fluid. The whole-cell patch clamp technology is used for recording the spontaneous action potential of the myocardial cells. The patch clamp amplifier adopts a 700B amplifier. The glass microelectrode is filled with electrode liquid, the electrode resistance used when recording action potential is 3-5M omega, and high resistance (-5G omega) sealing is formed between the electrode and the cell membrane. And (3) absorbing and breaking the cell membrane by using negative pressure, adjusting capacitance and series resistance compensation, wherein the sampling frequency is l0k Hz, the low-pass filtering frequency is 2k Hz, and recording the spontaneously-beating myocardial cell action potential after the electrode internal liquid and the intracellular liquid are balanced for 5min. The data were analyzed using pClamP 10.2 and Lab Chart 8.0 software.

The result shows that the action potential time course (APDs) of HCM-iPSC-CMs carrying MT-RNR2 mutation is obviously longer than that of the wild type; upon DNJ treatment, APDs were significantly shortened (fig. 4a 4 b. DNJ was shown to be able to improve the electrophysiological status of mutant cardiomyocytes.

Example 5

DNJ is coupled on magnetic beads, and an in-vitro pull-down experiment of cell lysate is carried out to verify a drug target.

Preparation of DNJ-coupled carboxyl magnetic beads: the experimental procedures were as per the product description of the PuRui Mige company. That is, the 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: 293T overexpressing EGFP and OPA1 was collected, lysed, the supernatant was incubated with beads and DNJ-beads for 4h at 4 ℃ and washed three times with lysis buffer, mixed with 40. Mu.L SDS loading buffer and boiled for 10min, and binding of EGFP and OPA1 to DNJ-beads was analyzed by Western blotting.

The results are shown in fig. 5, DNJ is able to bind overexpressed OPA1 in vitro, whereas the negative control EGFP is not. Indicating that there is an interaction between DNJ and OPA 1.

Example 6

The HCM-iPSC-CMs carrying the MT-RNR2 mutation obtained in example 1 were treated with DNJ and tested for multimer events on OPA 1.

Extracting mitochondria from the induced myocardial cells, adding 100 mu L KPBS containing 10mmol/L EDC for resuspension, standing at room temperature for 30min, adding 15mmol/L DTT to terminate the reaction, cracking mitochondria and extracting protein. And finally, obtaining a result through western blotting.

The results are shown in fig. 6, where DNJ treatment significantly increased multimer formation of mutant cardiomyocyte OPA 1. Indicating that DNJ promotes the function of OPA1 multimer formation by targeting OPA 1.

Example 7

The HCM-iPSC-CMs carrying the MT-RNR2 mutation obtained in example 1 were treated with DNJ and observed for morphology of the mitochondrial spine.

Cells were washed once with PBS and collected with a cell scraper. Add 1mL DPBS for resuspension, 3000rpm, centrifuge 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. Centrifuge at 3000rpm for 5min. Glutaraldehyde is removed as much as possible, and the solution is rinsed 2 times with 500. Mu.L of 0.1M PBS, each time at 4 deg.C for 10-15min. PBS was aspirated, cells were blown apart by addition of 1% osmic acid and fixed at 4 ℃ for 1h. Centrifuge at 3000rpm for 5min. Add 500. Mu.L ddH ₂ Rinse 2 times for 15min each time with O. Dyeing with 2% uranium acetate water solution, and standing at 4 deg.C for 30min. Gradient dehydration: adding 50%, 70%, and 90% alcohol in sequence for dehydration, and standing at 4 deg.C for 10min respectively. Dehydrating with 100% ethanol for 20min.100% acetone is dehydrated for 2 times, 20 min/time. The sample was mixed with anhydrous acetone and embedding medium at volume ratio of 1. Embedding with pure embedding medium, and polymerizing in an oven. 24 hours at 37 ℃; 24h at 45 ℃;68 ℃ and 48h. Polymerization in an oven: 24 hours at 37 ℃; 24 hours at 45 ℃;68 ℃ for 48h. And (3) staining the section: slicing with an ultrathin slicer, staining with 4% uranium acetate for 20min, and staining with lead citrate for 5min. And observing the sample by an electron microscope.

The results are shown in FIG. 7, and DNJ treatment promotes the mitochondrial ultrastructure of HCM-iPSC-CMs to be more ordered and the mitochondrial ridge to be more abundant and compact.

Example 8

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

One day before induction and differentiation of neural Stem cells, iPSCs are digested into single cells by Accutase (Stem cell), and then suspended in mTeSR1 (Stem cell) culture solution and cultured by 2.5-3.0 × 10 ⁵ Individual cells were uniformly seeded on Matrigel (BD) plated six-well plates. On day 0, when the cell density reached 10-20%, the medium was changed to neural stem induction medium (Gibico, cat. No. a 1647801) for 6 days with every two days. The neural stem cells of the P0 generation were obtained on day 7 and subjected to cell passage. Digesting the neural stem cells by using Accutase, then suspending the neural stem cells in a neural stem cell amplification medium, and adding 1.5 multiplied by 10 ⁶ Cells were seeded in Geltrex (Gibco, cat. No. a1413302) was plated onto six-well plates, and thereafter, the culture was continued with neural stem cell expansion medium with changing the medium every two days. When the confluence reaches 80% -90%, passage and frozen storage of the neural stem cells can be carried out. Neural stem cell induction medium includes neural basal medium and neural inducing supplement (50 ×). The neural stem cell amplification culture medium comprises a neural basal culture medium: DMEM/F12 (Gibco, cat # 11330-057) =1 and neuro-induced supplements (50 ×).

One day before neuron induction differentiation, digesting neural stem cells with Accutase, suspending the neural stem cells in neural stem cell culture medium, and adding 1 × 10 ⁵ Cells were seeded in lamin (10. Mu.g/mL) (Gibco, cat. No. 23017-015) plated six-well plates. On day 0, auditory neuron differentiation medium was added, after which the fluid was changed every other day. The neuron differentiation medium comprises a neural basal medium (Gibico, cat. No. 21103049), B-27 (50 x) (Gibico, cat. No. A3582801), glutamine (GlutaMAX) (100 x) (Gibico, cat. No. 35050061), a non-essential amino acid (NEAA) (100 x) (Gibico, cat. No. 11140050), and 20ng/mL brain-derived neurotrophic factor (BDNF) (R) of&No.248-BDB, cat), glial cell line-derived neurotrophic factor (GDNF) (R) 20ng/ml&D, cat. No. 212-GD) and 200. Mu.g/mL of L-Ascorbic Acid (AA) (Sigma-Aldrich, cat. No. 50-81-7). After 14 days of culture, experiments can be performed.

The cells were seeded by plating a slide in 24 wells. After washing with PBS for three times, 4% paraformaldehyde was added and fixed at room temperature for 15min. Then adding 0.2% Triton-X100, and permeating for 15min at room temperature. Then, 3% BSA was added and blocked at room temperature for 1h. TrkB (Abcam, cat # ab 18987), TUJ1 (Abcam, cat # ab 7751), neuN (Abcam, cat # ab 177487) were added overnight at 4 ℃. Then adding a fluorescent secondary antibody (the secondary antibody is Goat Anti-Rabbit IgG H)&L(Alexa

488 (Abcam)), incubated for 1h at room temperature in the absence of light. Further, 0.5mL of 1. Mu.g/mL DAPI was added, and the mixture was left at room temperature in the dark for 5min and sealed with 50% glycerol. During which each step was washed 3 times with PBS. And finally observing the result under a confocal microscope. The results are shown in FIG. 8, and the immunofluorescence results show that the induced neurons were homozygoticThe neuron marker proteins TrkB, neuN and TUJ1 are expressed successfully. The steps show that the iPSCs carrying the MT-RNR1 mutation can be successfully differentiated into neurons.

Example 9

The neurons carrying the MT-RNR1 mutation obtained in example 8 were treated with DNJ and subjected to electrophysiological examination.

The cell slide was plated in a 24-well plate and incubated with lamin for 1h. Neuronal cells were digested with Accutase, centrifuged and the supernatant discarded. Spreading the cells on a 24-well plate, 37 ℃,5% ₂ After 16 hours of incubation, the solution was changed, and the experimental group was added with a final concentration of 30. Mu.g/mL drug, and the control group was added with an equal volume of DMSO. The cultivation was continued for 48H. Taking out the cell slide, taking out the cell slide from the culture dish, placing the cell slide in an inverted microscope groove with a constant-temperature perfusion groove at 37 ℃, and continuously perfusing with extracellular fluid. And (3) drawing the glass microelectrode to an opening of about 1 mu M by using an electrode drawing instrument, filling about 1/3 of the volume of the electrode solution, installing the microelectrode to a recording probe, and filling the electrode solution to obtain a solution inlet resistance of 6-8M omega. After a positive pressure of 10mmHg was applied to the microelectrode, the micromanipulation was adjusted to be close to the cell. When the electrode approaches the cell and the electrode resistance is increased, removing the positive pressure, regulating the cell clamping voltage to-10 mV, simultaneously adding a smaller negative pressure to the cell, and removing the negative pressure after forming high-resistance sealing. Clamping the cells at-70 mV after sealing is stable, rapidly giving larger negative pressure and increasing an electric shock rupture membrane to form a whole-cell sealing state, and selecting the cells with membrane potential near-65 mV to record in a current clamp mode. In the current clamp mode, the cells were perfused with a current of 0-90pA with a current amplification of 10pA, and the response of the cells to depolarizing current was recorded.

The results showed that 48h after DNJ treatment of the neurons carrying MT-RNR1 mutations, the action potential depolarization current threshold decreased, and the action potential morphology improved under 90pA current stimulation, as represented by increased action potential amplitude, decreased rise time, and decreased time course (FIG. 9A, 9B). Indicating that DNJ significantly improved the function of mutant auditory neurons.

Claims

1. Use of compound DNJ for the preparation of a medicament for promoting the formation of OPA1 dimers.

2. Use of compound DNJ for the manufacture of a medicament for the treatment of a disease associated with an imbalance in OPA1 dimer formation.

3. The use according to claim 2, wherein the disorder associated with an imbalance in OPA1 dimer formation is hypertrophic cardiomyopathy, deafness or optic atrophy.

4. The use according to claim 3, wherein the hypertrophic cardiomyopathy results from a mutation in the MT-RNR2 gene.

5. The use according to claim 3, wherein the deafness is caused by a mutation in the MT-RNR1 gene.

6. The use of claim 4 or 5, wherein the mutation in the MT-RNR2 gene or MT-RNR1 gene causes mitochondrial dysfunction.

7. Use according to claim 1 or 2, wherein DNJ is used in an amount of 10 to 100 μmol/L.

8. A medicament for treating a disease associated with imbalance in OPA1 dimer formation, characterized in that the active ingredient is compound DNJ.

9. The medicament according to claim 8, wherein the disease associated with an imbalance in OPA1 dimer formation is hypertrophic cardiomyopathy, deafness, or optic atrophy.

10. The medicament of claim 9, wherein the hypertrophic cardiomyopathy is caused by a mutation in the MT-RNR2 gene; the deafness is caused by MT-RNR1 gene mutation.