Application of NOTCH pathway ligand JAG1 in regulation and control of cochlear hair cell regeneration

Abstract

The invention discloses application of a NOTCH pathway ligand JAG1 in regulation and control of cochlea hair cell regeneration, and relates to the technical field of cochlea hair cell regeneration. According to the invention, JAG1 in cochlea supporting cells plays a regulating role through a NOTCH1 / 2 receptor, and NOTCH3 does not play a role; in addition, PI3K-Akt-mTOR signal channels in cochlea supporting cells can be reduced due to deficiency of the P2 mouse JAG1, so that the proliferation level of the supporting cells is reduced, and the supporting cells are further dedifferentiated into hair cells; meanwhile, it is proved that the high-level JAG1 / Notch can promote the proliferation level of cochlea supporting cells; the invention also discloses that the expression of the JAG1 is reduced along with the maturation of the supporting cells after the mouse is born, which indicates that the JAG1 is associated with the plasticity of the supporting cells. Most importantly, the research finds that overexpression of JAG1 / ATOH1 in P2 mouse cochlea supporting cells can promote hair cell formation.

Description

Technical Field

[0001] The present invention relates to the technical field of cochlear hair cell regeneration, and in particular to an application of NOTCH pathway ligand JAG1 in regulating the regeneration of cochlear hair cells. Background Art

[0002] Deafness and hearing impairment are major global health issues. The ear is an important organ of hearing and balance for humans. Inner ear hair cells are key sensory cells for auditory conversion, which can convert sound into electrical stimulation transmitted to the brain. Their damage or loss is one of the main causes of deafness and hearing loss.

[0003] The auditory system consists of the auditory organ, auditory nerve, and auditory center. The integrity of the structure and function of the auditory organ is crucial to the formation of hearing. In mammals, cochlear hair cells are only produced during the developmental stage. The loss of cochlear hair cells due to genetic or environmental reasons is the main cause of deafness. However, in non-mammalian vertebrates such as birds and fish, hair cell damage will induce adjacent supporting cells to directly transdifferentiate into hair cells or re-enter the cell cycle to divide and form new hair cells and supporting cells. The cochlear supporting cells of adult mammals lack the ability to regenerate hair cells, but the cochlear supporting cells of perinatal mice still maintain a certain degree of regenerative stemness. In the immature cochlea of ​​perinatal mice, supporting cells can regenerate new hair cells as progenitor cells, but this regenerative ability is lost as they mature. Summary of the invention

[0004] The present invention provides an application of a NOTCH pathway ligand JAG1 in regulating the regeneration of cochlear hair cells. JAG1 is used to activate the NOTCH signaling pathway, and it is found that JAG1 can effectively induce supporting cells to transform into new hair cells, thereby improving or restoring hearing function.

[0005] To achieve the above object, the present invention provides an application of a NOTCH pathway ligand JAG1 in regulating the regeneration of cochlear hair cells, wherein the CDS sequence of the JAG1 is shown in SEQ ID NO.1.

[0006] Preferably, the regulating cochlear hair cell regeneration comprises:

[0007] (1) JAG1 deficiency increases hair cell formation in cochlear tissue and organoids;

[0008] (2) JAG1 deficiency downregulates the PI3K-AKT-mTOR signaling pathway in cochlear supporting cells, leading to a decrease in the proliferation level of supporting cells and further differentiation into hair cells;

[0009] (3) Stimulation of JAG1 signaling enhances the ability of mature cochlear supporting cells to form hair cells in an mTOR-dependent manner.

[0010] The use of a NOTCH pathway ligand JAG1 in regulating the regeneration of cochlear hair cells of the present invention has the following beneficial effects:

[0011] (1) published that JAG1 deficiency in P2 mice increased hair cell formation in cochlear tissue and organoids;

[0012] (2) demonstrated that JAG1 deficiency reduced the expression of key progenitor and metabolic genes and attenuated PI3K-AKT-mTOR signaling in cochlear supporting cells and Crickell's cells;

[0013] (3) It was demonstrated that increasing JAG1 / NOTCH signaling can enhance cochlear supporting cells / ker cells' mitotic capacity;

[0014] (4) revealed that JAG1 expression decreases with the maturation of supporting cells and that stimulation of JAG1 signaling enhances the ability of mature cochlear supporting cells to form cochlear hair cells in an mTOR-dependent manner;

[0015] (5) Using JAG1 as a ligand of the NOTCH signaling pathway to stimulate the transformation of cochlear supporting cells into hair cells, thereby promoting the regeneration of hair cells, is of great significance for the treatment of hearing loss caused by hair cell damage or loss;

[0016] (6) The present invention not only provides a new idea for the treatment of sensorineural hearing loss, but also helps to deepen the understanding of the cell regeneration mechanism in the cochlea, laying the foundation for the development of more effective hearing loss treatment methods in the future.

[0017] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The results of functional detection of JAG1 gene during organoid culture; Part A is the P2 organoid culture process; Part B is the confocal images of JAG1 and Hoechst in the control and Jag1 cKO organoids; Part C is the RT-PCR of Jagl expression in the control and Jagl cKO organoids on the 7th day of expansion; Part D is the white light image of the control and Jagl cKO organoids at 7 days of expansion; Part E is the corresponding organoid formation efficiency in Part D (the figure shows individual data points and mean ± SD, n = 8 animals in each group); Part F is the corresponding organoid diameter in Part D (the figure shows individual data points and mean ± SD, n = 8 animals in each group); Part G is the cell proliferation of the control and Jagl cKO organoids at 7 days of expansion culture; Part H is the corresponding EdU in Part G +Cell percentage (the figure shows individual data points and mean ± SD, n = 6 animals per group); Part I shows RT-PCR of Notch target gene Hesl, supporting cell markers and hair cell markers in control and Jagl cKO organoids after 7 days of expansion; Part J shows RT-PCR of hair cell markers in control and Jag1 cKO organoids after 5 days of differentiation (mean ± SD, n = 3 mice / group); P values ​​were calculated using two-tailed unpaired t-test, *P < 0.05, **P < 0.01, ***P < 0.001;

[0019] Figure 2 Expression analysis of classical Notch target genes and effector genes in Jag1 cKO organoids and control organoids; two-tailed, unpaired Student's t-test was used to calculate P values, *P≤0.05, **P<0.01, ***P<0.001 and ****P<0.0001; Part a is the experimental schematic diagram; Part b is the volcano plot of RNA sequencing data in response to Jag1 cKO; Part c is the biological processes and pathways related to Jag1 cKO downregulated genes, sorted by corrected P value (q value); Part d is Jag1, Hey1, Id1, Eif4ebp1, Minar2, Nu RT-PCR for mRNA expression of pr1, Dkk3, and Scx (n=4); Part e is immunoblotting for p-AKT, AKT, p-S6, p-4EBP1, 4EBP1, JAG1, and β-actin in organoids; Part f is quantification of p-AKT, p-S6, and p-4EBP1 proteins in part e (n=3, from one representative);

[0020] Figure 3 This is a P4 cochlear explant proliferation test; Part A is a schematic diagram of the P4 cochlear explant proliferation test; Part B is a confocal image of the hair cell layer of the cochlear explant after 5 days of culture; Part C is the corresponding hair cell formation in Part B (MYO7A + Ato h1-nGFP + SOX2 + ) quantitative analysis. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and advantages disclosed in the embodiments of the present invention more clearly understood, the embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the embodiments of the present invention and are not intended to limit the embodiments of the present invention. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0022] Embodiment 1

[0023] The method for promoting the regeneration of cochlear hair cells by activating the NOTCH signaling pathway by JAG1 comprises the following steps:

[0024] S1. Cochlear organoid culture

[0025] The regenerative capacity of cochlear hair cells in perinatal mice gradually decreases with development and maturation, and cochlear organoids were cultured from 2-day-old newborn mice.

[0026] The cochlea was dissected in a sterile PBS solution, and the cochlear basement membrane was obtained. The cochlear basement membrane was digested with dispase and collagenase at a final concentration of 1 mg / mL at room temperature for 8 minutes, and then the digestion was terminated with DMEM / F12 solution containing 10% fetal bovine serum.

[0027] After standing at room temperature for 30 minutes, the cochlear epithelium was separated using a disposable injection needle, placed in a 1.5 mL centrifuge tube, and incubated at 37°C for 20 minutes with trypsin (TrypLE) solution.

[0028] After terminating digestion and blowing off cells, filter the cell suspension through a 35μm filter to obtain a single cell suspension. Take an equal amount of cell suspension and mix it with Matrigel 1 (Matrigel) at a ratio of 1:1, and drop it into a preheated four-well plate. Place it in a 37°C incubator for 20 minutes. After the Matrigel is completely solidified, add culture medium to each culture well to immerse the Matrigel.

[0029] Cochlear organoids proliferated in proliferation medium [complete medium (DMEM / F12), N-2 supplement (N-2supplement), B-27 supplement (B-27supplement), epidermal growth factor (EGF), fibroblast growth factor (FGF-2), GSK-3α / β inhibitor (CHIR99021), HDAC inhibitor (VPA), selective TGFβR-1 / ALK5 inhibitor (616452), penicillin] for 10 days, and then changed to differentiation medium [complete medium (DMEM / F12), N-2 supplement (N-2supplement), B-27 supplement (B-27supplement), GSK-3α / β inhibitor (CHIR99021), γ-secretase inhibitor (LY411575)] for 10 days.

[0030] Atoh1-nGFP (MGI: 3703598) transgenic mice can specifically mark cochlear hair cells with GFP green fluorescence. When this mouse is used to culture cochlear organoids, the formation of hair cells can be indicated by green fluorescence during the differentiation stage.

[0031] S2, JAG1 gene

[0032] The JAG1 genotype mice were introduced into the Atoh1-nGFP genotype by hybridization to label hair cells.

[0033] The CDS sequence of JAG1 is shown in SEQ ID NO.1.

[0034] To conditionally knock out (cKO) Jag1, Jag1 cKO (Tet-on-Cr e tg / +; M2rtTA tg / +; Jag1 f / f ) mice and controls (M2rtTA tg / +; Jag1 f / f ) cochlear epithelial cells of the same littermates were used to culture cochlear organoids, and gentamicin (dox) was added when plating and replenished every other day. Figure 1 As shown in part A, cochlear organoids proliferated in proliferation medium [complete medium (DMEM / F12), N-2 supplement (N-2sup plement), B-27 supplement (B-27supplement), epidermal growth factor (EGF), fibroblast growth factor (FGF-2), GSK-3α / β inhibitor (CHIR99021), HDAC inhibitor (VPA), selective TGFβR-1 / ALK5 inhibitor (616452), penicillin] for 11 days, and then changed to differentiation medium [complete medium (DMEM / F12), N-2 supplement (N-2supplement), B-27 supplement (B-27supplement), GSK-3α / β inhibitor (CHIR99021), γ-secretase inhibitor (LY411575)] for 5 days of differentiation.

[0035] Confocal images of JAG1 (magenta) and Hoechst (blue) in control and Jag1 cKO organoids. Figure 1 As shown in part B, scale bar = 50 μm. The results showed that cells in the control group organoids highly expressed JAG1 protein on their cell surface, while cells in the Jag1 cKO group organoids lacked JAG1 protein.

[0036] RT-PCR of Jagl expression in control and Jagl cKO organoids on day 7 of expansion. Figure 1 As shown in Part C (mean ± SD, n = 3 mice / group). The results showed that the abundance of Jagl transcripts in Jagl cKO cultures was 1000-fold lower than that in control cultures, confirming the successful deletion of JAG1.

[0037] White light images of control and Jagl cKO organoids at 7 days of expansion. Figure 1 Shown in middle part D. Scale bar = 400 μm.

[0038] After 7 days of expansion, colony formation efficiency (number of organoids per input cells) and organoid growth (organoid diameter) were analyzed in control and Jagl cKO cultures.

[0039] Colony formation efficiency Figure 1 As shown in Part E, the results show that the colony formation efficiency in the control culture was approximately 2-fold higher than that in the Jag1 cKO culture.

[0040] The diameter of the organoid Figure 1 As shown in part F, the results show that the diameter of organoids in control cultures is approximately 1.5 times larger than that in Jag1 cKO cultures.

[0041] A single EdU pulse was given at 7 days of expansion, and EdU incorporation (green) and Hoechst-labeled nuclei (blue) were analyzed 1 hour later. Cell proliferation in control and Jagl cKO organoids is shown in Figure 2. Figure 1 As shown in part G, EdU + The percentage of cells Figure 1 As shown in Figure 2, the smaller organoid size observed in Jag1 cKO cultures was due to a 1.6-fold lower cell proliferation rate.

[0042] In summary, the above findings suggest that JAG1 deficiency attenuates cochlear supporting cells and Mitotic potential of iker cells.

[0043] Figure 1 Figure 1. RT-PCR analysis of Notch target gene Hes1, supporting cell markers (Fgf20, Sox2, S100a1, Lfng) and hair cell markers (Atoh1, Pou4f3, Gfi1, Myo7a) in control and Jag1 cKO organoids at 7 days of expansion. Loss of Jag1 reduced the expression of genes critical for foresensory development (Fgf20, Sox2) and hair cell 131 fate inhibition (Hes1).

[0044] Figure 1 RT-PCR detection of hair cell markers (Atoh1, Pou4f3, Myo7a) in control and Jag1 cKO organoids after 5 days of differentiation was analyzed in J, indicating that the rate of hair cell generation was higher in Jag1 cKO organoid cultures compared to control cultures.

[0045] S3 and JAG1 in SC supporting cells and Function in cells

[0046] Jag1 cKO (Tet-on-Cre tg / +; M2rtTA tg / +; Jag1 f / f ) mice and controls (M2rtTAtg / +; Jag1 f / f ) littermates, cochlear sensory epithelial cells were isolated, and cochlear organoids were cultured under dox-induced Jag1 deficiency, as described in Figure 2 As shown in part a.

[0047] To gain further insights into the role of JAG1 in SC and Function in cells, gene expression in control and Jag1 cKO organoids after 7 days of expansion was analyzed using RNA sequencing (RNA-seq). A total of 802 differentially expressed genes (DEGs) were identified (q value 0.01), 160 genes were upregulated and 642 genes were downregulated in Jag1 cKO organoids compared with control organoids. Consistent with the key role of JAG1 in classical Notch signaling. It was found that Jag1 cKO organoids expressed classical Notch target genes and effector genes (Nrarp1, Sox2, Hes1, Hey1 and HeyL) at significantly lower levels than control organoids, such as Figure 2 As shown in parts b and d.

[0048] like Figure 2 As shown in middle part (b), among the gene sets significantly upregulated in Jag1 cKO organoids compared with controls are Scx and Smo c2, which are expressed in supporting cells and non-sensory epithelial cells, and supporting cell-specific genes Tesc and Pou4f3.

[0049] To identify pathways and biological processes that may be disrupted by the loss of JAG1-mediated signaling, the list of downregulated genes was subjected to gene ontology enrichment analysis using the web-based portal Metascape. Figure 2 As shown in part c, the results showed that “Notch signaling pathway” and “regulation of nervous system development” were the top 20 pathways and biological processes.

[0050] To determine whether PI3K-Akt-mTOR signaling is downregulated in Jag1 cKO organoids, the expression levels of the phosphorylated (p) form of the kinase Akt (p-Akt, Ser473), ribosomal protein S6 (p-S6, Ser240 / 244), and eukaryotic initiation factor 4E binding protein 1 (p-4EBP1, Thr37 / 46) proteins were analyzed by immunoblotting, and the results are shown in Table 1. Figure 2 As shown in e and f.

[0051] Increased protein levels of p-S6 (Ser240 / 244) and p-4EBP1 (Thr37 / 46) correlate with increased mTOR activity, while p-Akt (Ser473) is a readout of maximal Akt activation downstream of PI3K. Analysis revealed that protein levels of p-AKT and p-4EBP1 were significantly reduced in Jag1 cKO organoids compared to controls, identifying JAG1 as a positive regulator of PI3K-AKT-mTOR signaling.

[0052] S4. Cochlear explant culture

[0053] First, the mouse cochlea was dissected in a sterile PBS solution, the basilar membrane was kept wrapped around the modiolus, the bottom end of the modiolus was trimmed flat with forceps, and finally it was placed on the SPI-Pore membrane for suspension culture.

[0054] Cochlear sensory epithelial cells were isolated from newborn 4-day-old mice by microdissection, sampled and established as cochlear explants, and cultured on SPI-Pore membrane filters (Structure Probe, No. E1013-MB) in DME M / F12 containing 1×N-2 supplement, epidermal growth factor (5 ng / mL) and penicillin (100 U / mL; Sigma-Aldrich, No. P3032). To induce Tet-On-Cre transgene expression, 0.5 μg / mL doxycycline was added to the culture medium. Gentomycin sulfate (100 μg / mL; Sigma-Aldrich, No. G1272) was used to ablate HCs.

[0055] Figure 3 Part A is a schematic diagram of cochlear explant culture, where cochlear explants obtained from P4 Atoh1-nGF P transgenic mice were cultured in the presence of JAG1 peptide or BSA (control) and rapamycin or DMSO (vehicle control). On the next day, Wnt activator (CHIR 99021) and Notch inhibitor (LY411575) were added to induce hair cell formation.

[0056] Cochlear explants were suspended in culture medium (DMEM / F12, N2 supplement, B-27 supplement, CHIR99021, LY411575), and Ed U reagent was added during the culture process to mark proliferating cells. After 5 days of culture, the explants were collected and analyzed by immunofluorescence staining and imaged under a laser scanning confocal microscope.

[0057] S5. EdU proliferation assay

[0058] EdU reagent is added to the organoid culture medium to mark proliferating cells. The cell proliferation level is detected after EdU staining. EdU is added to the cochlear explant culture medium, and the regeneration mode of new hair cells formed from supporting cells is detected based on the EdU staining results.

[0059] Among them, Sox2 + Myo7a + The hair cells are derived from direct transdifferentiation of supporting cells, while Sox2 + Myo7a + EDU + Hair cells are formed by supporting cells re-entering the cell cycle and undergoing transdifferentiation.

[0060] S6. Immunofluorescence staining

[0061] Myo7a antibody was used to label hair cells, Sox2 antibody was used to label supporting cells, and EdU staining was used to label proliferating cells and indicate the origin of newborn hair cells.

[0062] Among them, before staining, the organoid cell suspension was mixed with Matrigel 1 (Matrigel), and then re-plated on the coverslip. Subsequent staining was performed after the Matrigel 1 solidified. During the staining process of cochlear explants, the explants were retained on the silver filter (SPI-Pore) membrane. After the staining was completed, the explants were transferred from the membrane to the slide with a disposable syringe and then sealed. All immunofluorescence staining samples were imaged under a laser scanning confocal microscope.

[0063] like Figure 3 Part B shows a confocal image of the hair cell layer of a cochlear explant (middle apical turn) after 5 days of culture. Myo7a (magenta), Atoh1-nGFP (green), and Sox2 (blue) mark newly formed hair cells, and EdU (red) marks proliferating cells.

[0064] Quantitative analysis of newly formed hair cells (MYO7A+SOX2+Atoh1-nGFP+) was performed, as Figure 3 As shown in middle C, analysis revealed that rapamycin significantly reduced the number of hair cells (MYO7A+Atoh1-nGFP+SOX2+) formed in JAG1-treated cultures, indicating that the JAG1 peptide stimulates new hair cell formation at least in part by activating mTOR signaling.

[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A use of NOTCH pathway ligand JAG1 in regulating cochlear hair cell regeneration, characterized in that: The CDS sequence of JAG1 is shown in SEQ ID NO.

1.

2. The use of a NOTCH pathway ligand JAG1 in regulating cochlear hair cell regeneration according to claim 1, characterized in that: The regulating cochlear hair cell regeneration comprises: (1) JAG1 deficiency increases hair cell formation in cochlear tissue and organoids; (2) JAG1 deficiency downregulates the PI3K-Akt-mTOR signaling pathway in cochlear supporting cells, leading to a decrease in the proliferation level of supporting cells and further differentiation into hair cells; (3) Stimulation of JAG1 signaling enhances the ability of mature cochlear supporting cells to form hair cells in an mTOR-dependent manner.

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