Rps14 upregulation promotes inner ear progenitor proliferation and hair cell regeneration in the neonatal mouse cochlea

Abstract Sensorineural hearing loss a result from hair cell damage, which is irreversible in mammals owing to the lack of hair cell regeneration, but recent researches have shown that Lgr5 + supporting cells are progenitors capable of regenerating hair cells. RPS14 (ribosomal protein S14) is a 40S ribosomal subunit component and is associated with erythrocyte differentiation, and in this study, we used a novel adeno‐associated virus‐inner ear system to upregulate Rps14 expression in cultured hair cell progenitors and observed an enhancement on their ability to proliferate and differentiate into hair cells. Similarly, Rps14 overexpression in the mice cochlea could promote supporting cells proliferation by activating the Wnt signalling pathway. In addition, over‐expressing Rps14 induced hair cells regeneration in the organ of Corti, and lineage tracing showed that the new hair cells had transformed from Lgr5+ progenitors. In conclusion, our analysis reveals the potential role of Rps14 in driving hair cell regeneration in mammalian.


| INTRODUCTION
Hearing loss affects 6%-8% of the global population. 1 The hair cells located in the inner ear cochlea are highly specialized mechanoreceptor cells that are responsible for sound detection and signal transmission. In most sensorineural hearing loss genetic abnormalities, excessive noise, ototoxins, infections, ear infections and aging, 2 auditory hair cells undergo apoptosis and are permanently lost in mammals because of the inability to regenerate new hair cells. 3,4 However, in non-mammalian vertebrates such as birds, supporting cells are can spontaneously generate new hair cells in the auditory sensory epithelia throughout the animal's entire lifespan. 5 In mammals, auditory hair cells are only differentiated naturally during the course of embryonic development, 6 and studies have shown that inner ear supporting cells have the properties of hair cell progenitors and can regenerate hair cells after injury only during the newborn period. [7][8][9] Hair cells and supporting cells have a close lineage relationship during auditory epithelia development, [10][11][12] and supporting cells in the murine cochlea Lgr5 +7 or Frizzled9 +13 along with the greater epithelial ridge, 14 can act as progenitors to produce neo-hair cells through direct transdifferentiation ormitotic regeneration.
In the murine cochlea, multiple signalling pathways regulate the trans-differentiation of supporting cells into hair cells. The reactivation of the transcriptional activator Atoh1 in supporting cells is an indispensable step during hair cell regeneration, 15,16 and Atoh1 is also essential for the development of later hair cell in the embryonic stage. 17,18 Continuous activation of Wnt/β-catenin facilitates the proliferation and differentiation of Sox2 + progenitors and is also necessary for new hair cell formation. 19 Similarly, in damaged cochleae, supporting cells produce neo-hair cells by inhibiting Notch signalling, 20 and overexpression of LIN28 enables new hair cell production from supporting cells through mitotic and nonmitotic mechanisms. Finally, the mutual antagonists Lin28b and Let-7 regulate the regenerative capacity of supporting cells in an mTORC1-dependent manner. 21,22 RPS14 (ribosomal protein S14), encoded by the Rps14 gene, is a the 40S ribosomal subunit section and is considered to an indispensable component of ribosomal biogenesis. In Rps14 haploinsufficient cells, the S100 and p53 proteins are involved in the activation of a self-regulating feedback loop that leads to a block in terminal erythroid differentiation, 23 thus suggesting a role for Rps14 in erythrocyte differentiation. Furthermore, studies have shown that in oestrogen receptor (ER)-positive breast cancer tissues, RPS14 is highly expressed compared with ER-negative breast cancer and that reducing the expression of Rps14 inhibits cell proliferation and metastasis. 24 Our data showed that in neonatal mice Rps14 is expressed in the cochlear epithelia with decreased expression as the animal ages, suggesting that Rps14 might be involved in the development and maturation of the sensory epithelia as well as in the regeneration ability of supporting cell progenitors. Therefore, we investigated the effect of Rps14 regulation on the proliferation and differentiation abilities of supporting cells using recombinant adeno-associated virus (AAV). Several gene delivery systems have been tested for use in cochlear supporting cells, including AAV, adenovirus, lentivirus, herpes simplex virus and transfection of antisense oligonucleotides. 25 The ability of AAV to efficiently infect non-dividing cells makes it particularly valuable for gene delivery. In particular, a recent study showed that a new AAV variant named AAV-ie (AAV-inner ear) can transduce nearly 90% of the supporting cells through round window membrane injection and that AAV-ie-mediated Atoh1 transfer induces significant transdifferentiation of supporting cells into neo-hair cells. 26 So, AAV-ie is capable of facilitating the exogenous re-expression of Rps14 in cochlear progenitors derived from supporting cells.
Here, we simulated cell division and hair cell differentiation in cultured organoids derived from supporting cells in both twodimensional and three-dimensional systems, and we found that the overexpression of Rps14 increased the organoid-forming ability of supporting cells and promoted hair cell differentiation. quantitative real-time polymerase chain reaction (QPCR) results showed that

| AAV injection through the round window membrane
Neonatal mice (P1-2) were anaesthetised on ice for 2-3 min and then moved to an ice pad for subsequent surgery. The surgery was performed on the left ear of each animal within 5-10 min of each control. After anaesthesia, the otic bulla was carefully exposed and the round window membrane was observed. Special attention was paid to avoid injury to the facial nerve. The injection was performed via a glass pipette with a 25-mm tip (Drummond, #5-000-1001-X10) controlled by a UMP3 ultra micro pump (World Precision Instruments). The total volume of AAVs for injection was about 1.5 μL per cochlea. Following virus injection, surgical wounds were glued using veterinary tissue adhesive (Millpledge Veterinary, #LMIL135). The mouse pups were then placed on a warm plate at 38 C for 10 min for recovery before being returned to their mothers for continued nursing.

| Immunostaining
Cochleae were dissected and fixed in 4% paraformaldehyde (PFA) at room temperature for 2 h. After fixation, the samples were decalcified   Table S1.

| Statistical analysis
Results are reported as mean ± SEM. Statistical analyses were performed by GraphPad Prism 9 (GraphPad Software, Inc.) with twotailed Student's t-test or one-way analysis of variance (ANOVA) with Tukey's multiple comparison test. p < 0.05 was considered statistically significant.

| AAV-Rps14 enhances the proliferation of hair cell progenitors in cochlear organoids
We first performed immunofluorescence staining to explore the sub-   (Figure 3L,O). The number of hair cells (myosin7a + cells) in each organoid was also increased ( Figure 3P). Similarly, we also observed an increase in the number of EdU + proliferating cells in Rps14-overexpressing organoids ( Figure 3L). Together these results suggest that Rps14 overexpression can enhance the ability of hair cell progenitors to differentiate into hair cells in vitro. Next, we also detected the downstream target genes of classic pathways involved in the hair cells regeneration in these two groups. Our QPCR analysis showed that the transcriptional expression of genes in Notch signalling pathway, like Dlk1, was significantly changed after Rps14 overexpression ( Figure 3Q). We also detected a decrease of FGF signalling receptor Fgfr4 in AAV-Rps14 transduced organoids ( Figure 3Q).
We also quantified the differentiation ability of AAV-Rps14 expanded progenitors. The virus were added at the beginning of the organoids expansion ( Figure 4A). After 5 days expression and 7 days differentiation, the average numbers of hair cells in each well and each organoid were counted, which showed significantly increases compared to the control groups ( Figure 4B-D). And, the ratio of myo-sin7a/DAPI is higher than those in the controls ( Figure 4B,E).
Together, these results demonstrate that Rps14 overexpression promotes the stemness of cultured progenitor cells in three-dimensional culture system.

| AAV-Rps14 promotes the proliferation of supporting cells in the mouse cochlea via wnt signalling pathway
In the rodent cochlea, terminally differentiated hair cells cannot spontaneously regenerate after injury. 33 In recent years, however, a series of studies have shown that supporting cells can act as inner ear progenitors for hair cell reproduction through the forced expression of the Wnt, Notch or Atoh1 signalling pathways. 10,14 Given that Rps14 is involved in the proliferation and differentiation behaviour in different eukaryotes, 23,24 we studied the effects of Rps14 overexpression in cochlear progenitor cells. We tested whether exogenous Rps14 overexpression is required for young, immature supporting cells to proliferation and produce hair cells in the mouse cochlea in situ. AAV-Rps14 and control virus were injected through the round window membrane at P1, and EdU was injected intraperitoneally (50 mg/kg body weight) from P2 to P4 to label proliferating supporting cells ( Figure 5A). Expression of Rps14 in supporting cells was confirmed by QPCR and immunofluorescence ( Figures 5A,B and S1). Both AAV-Rps14 and control AAVs effectively transduced supporting cells as we previously reported. 26 Mice were sacrificed and cochlear samples were collected at P7 for further EdU observation ( Figure 5A,C).
EdU + /Sox2 + supporting cells were detected in AAV-Rps14-injected cochleae, whereas no EdU + /Sox2 + supporting cells were observed in control cochleae ( Figure 5C,D), indicating that the proliferative ability of Rps14-overexpressing supporting cells was enhanced. The canonical Wnt/β-catenin signalling pathway regulates supporting cell proliferation during cochlear development, 34 and Lgr5 is the downstream target gene of Wnt signalling. 28,35 Lgr5-EGFP mice were used to The results are shown as the mean ± SEM. The p-value was calculated by Student's t-test (*p < 0.05; **p < 0.01; ***p < 0.001; n.s. refers to no significance.). AAV, adeno-associated virus; QPCR, quantitative real-time polymerase chain reaction greater numbers of hair cells. Thus, the reprogramming of hair cells in vivo was further explored via Rps14 upregulation. First, the cochleae of P2 wild-type mice were injected with the same numbers of AAV-Rps14 and control viruses through the round window membrane, and the cochlear samples were collected 7 days later for immunofluorescence observation ( Figure 6A). We observed that the heterogeneous hair cells were mainly concentrated at the site of the inner hair cells (IHCs), so our subsequent immunofluorescence images only showed the IHCs ( Figure S2). A small number of ectopic hair cells near the IHC region of the AAV-Rps14 groups could be detected at a dose of 4.5 Â 10 10 GCs, but this was not statistically different compared to the control cochleae ( Figure S3). This might be due to the lower expression level of exogenous Rps14 caused by an insufficient number of AAV-Rps14 virus particles. Thus, we doubled the number of AAV-Rps14 virus particles (9 Â 10 10 GCs), and as predicted the total number of ectopic IHCs in the cochlear spiral was significantly increased after the expression of exogenous Rps14 was increased at P9 ( Figure 6B,C). Specifically, the number of ectopic IHCs in the basal turns of AAV-Rps14-infected cochleae were significantly increased, while there was no obvious difference in the apical and middle turns of the cochlea between the AAV-Rps14 and the control groups ( Figure 6C). Furthermore, ectopic IHCs were still found at P16 in AAV-Rps14-infected cochleae ( Figure 6B), while the ectopic IHCs in the AAV-Rps14-infected cochleae were significantly reduced with no statistical significance compared with the control groups at the same age ( Figure 6D). These results indicated that the regenerated ectopic IHCs would undergo apoptosis over time.

| AAV-Rps14 effectively induced the Lgr5+ supporting cell to hair cells transformation
We next investigated the source of the increased extra hair cells.
Cochlear Lgr5 + progenitors are considered to be the source of regenerated hair cells in the cochlea of neonatal mouse, 7,29,39 including inner pillar cells, inner border cells and third-row Deiters' cells. 40 We syndrome gene, 44 and studies have shown that Rps14 haploinsufficiency leads to p53-dependent erythrocyte differentiation defects accompanied by apoptosis. 23 Rps14 has not been studied previously in the inner ear, and here we have over-expressed Rps14 in cochlear supporting cells using the AAV-ie system that targets supporting cells.
We found that overexpression of Rps14 also induced supporting cell trans-differentiation and thus increased the hair cell number in cul-  Tamoxifen was injected at P1, and AAV was injected 12 h later. Sox2 + supporting cells were traced by following the expression of tdTomato fluorescent protein (red). Cochlea were injected with AAV-mNeonGreen and AAV-Rps14 at the same dose (6 Â 10 10 GCs per cochlea) and harvested at P9. Myosin7a (cyan) marks hair cells. Scale bars, 50 μm. (C,E) Quantification of tdTomato + OHCs (C), IHCs (D) and hair cells (HCs) (E) per cochlea. Yellow arrowheads indicate the OHCs, and the triangles indicate the IHCs. n refers the number of mice, and the results are shown as the mean ± SEM (n = 4). The p-value was calculated by one-way ANOVA with Tukey's multiple comparison test. (*p < 0.05, **p < 0.01, ***p < 0.001). IHCs, inner hair cells; OHCs, outer hair cells.
In recent years, although the genes mentioned above have been shown to regulate the proliferation and differentiation of supporting cells and their differentiation into hair cells, the efficiency and application of hair cell regeneration for injury repair in the mammalian cochlea has been limited. New genes involved in cochlear repair need to be identified, and our study shows that Rps14 upregulation in cochlear supporting cells increases the number of hair cells by inducing supporting cell trans-differentiation, which suggests that Rps14 may be a new candidate gene for hair cell regeneration in the cochlea.
The combination of Rps14 with Wnt and Notch will be a promising strategy for therapeutic HCs regeneration.