Autophagy protects auditory hair cells against neomycin-induced damage

Abstract

Aminoglycosides are toxic to sensory hair cells (HCs). Macroautophagy/autophagy is an essential and highly conserved self-digestion pathway that plays important roles in the maintenance of cellular function and viability under stress. However, the role of autophagy in aminoglycoside-induced HC injury is unknown. Here, we first found that autophagy activity was significantly increased, including enhanced autophagosome-lysosome fusion, in both cochlear HCs and HEI-OC-1 cells after neomycin or gentamicin injury, suggesting that autophagy might be correlated with aminoglycoside-induced cell death. We then used rapamycin, an autophagy activator, to increase the autophagy activity and found that the ROS levels, apoptosis, and cell death were significantly decreased after neomycin or gentamicin injury. In contrast, treatment with the autophagy inhibitor 3-methyladenine (3-MA) or knockdown of autophagy-related (ATG) proteins resulted in reduced autophagy activity and significantly increased ROS levels, apoptosis, and cell death after neomycin or gentamicin injury. Finally, after neomycin injury, the antioxidant N-acetylcysteine could successfully prevent the increased apoptosis and HC loss induced by 3-MA treatment or ATG knockdown, suggesting that autophagy protects against neomycin-induced HC damage by inhibiting oxidative stress. We also found that the dysfunctional mitochondria were not eliminated by selective autophagy (mitophagy) in HEI-OC-1 cells after neomycin treatment, suggesting that autophagy might not directly target the damaged mitochondria for degradation. This study demonstrates that moderate ROS levels can promote autophagy to recycle damaged cellular constituents and maintain cellular homeostasis, while the induction of autophagy can inhibit apoptosis and protect the HCs by suppressing ROS accumulation after aminoglycoside injury.

Keywords: aminoglycosides; apoptosis; autophagic flux; autophagosome; hair cell protection; lysosome; oxidative stress.

Figure 1.

Increased autophagy in cochlear HCs after neomycin treatment. (A) Western blotting using total cochlear homogenates showed changes of LC3B-II expression in the cochleae treated with different concentrations of neomycin (0.2 mM, 0.5 mM, 1 mM, and 2 mM) and different exposure times (6 h and 24 h). GAPDH served as the sample loading control, n = 3. (B) Quantification of the western blot in (A). (C) Transmission electron microscope (TEM) analysis to evaluate autophagy in cochlear HCs. The numbers of autophagic vacuoles and autolysosomes (arrows in pictures) were significantly increased after neomycin treatment compared with the control, n = 3. (D) Quantification of the results in C. (E) Immunofluorescence staining with MYO7A antibody in the cochleae from GFP-LC3B mice. The GFP-LC3B puncta were significantly increased with neomycin treatment, n = 6. (F) Quantification of the GFP-LC3B punctum number in E. For all experiments, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2.

Increased autophagy in HEI-OC-1 cells after neomycin treatment. (A) Western blot showing the changes in LC3B-II expression in the HEI-OC-1 cells treated with different concentration of neomycin (0.5 mM, 1 mM, 2 mM, and 5 mM) and different exposure times (6 h and 24 h), n = 3. (B) Quantification of the western blot in (A). (C) TEM analysis to evaluate the presence of autophagy in HEI-OC-1 cells. The numbers of autophagic vacuoles and autolysosomes (arrows in pictures) were significantly increased after neomycin treatment compared with the control, n = 3. (D) Immunofluorescence staining with anti-LC3B antibody in HEI-OC-1 cells after neomycin injury, n = 4. (E) Quantification of the results in (C). (F) Quantification of the LC3B fluorescent puncta in (D). (G) Western blots with anti-LC3B antibody after neomycin and Baf treatment, n = 4. (H) Quantification of the western blot result in (G). (I) Immunofluorescence staining with anti-LC3B and anti-SQSTM1/p62 antibodies in HEI-OC-1 cells. The colocalization puncta (arrows in pictures) appeared after neomycin treatment. (J) Western blots with anti-SQSTM1/p62 antibody revealed a significant decrease in SQSTM1/p62 after neomycin treatment and significant increase after Baf treatment, n = 3. (K) Quantification of the western blot result in (J). (L) Transfection with mRFP-GFP-LC3B plasmids in HEI-OC-1 cells, n = 4. (M) Quantification of the results in (L). (N) HEI-OC-1 Cells were transfected with NHTT-150Q-EGFP and treated with neomycin, n = 4. For all experiments, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3.

Rapamycin and 3-MA affected both the induction of autophagy and HC survival in the cochlea after neomycin damage. (A) Immunofluorescence staining with the anti-MYO7A antibody in the cochleae from GFP-LC3B mice after different treatments, n = 6. (B) Quantification of the number of GFP-LC3B puncta in (A). The number of GFP-LC3B puncta was significantly increased in the rapamycin-pretreatment group and significantly decreased in the 3-MA-pretreatment group after neomycin treatment compared with neomycin treatment alone, n = 6. (C) Quantification of the MYO7A-positive HCs in Figure S4. Pretreatment with rapamycin promoted HC survival from the apical to the basal turn of the cochlea compared with neomycin exposure alone. In contrast, 3-MA accelerated HC apoptosis after neomycin injury, n = 6. Scale bars: 10 μm. For all experiments, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4.

Inhibition and activation of autophagy affect autophagosome formation and cell survival in HEI-OC-1 cells following neomycin damage. (A) Images of immunolabeled LC3B (green) in HEI-OC-1 cells, n = 4. (B) Quantification of the LC3B fluorescent puncta in (A). (C) Cells were transfected with mRFP-GFP-LC3B plasmids and treated with different drugs. Yellow dots indicate autophagosomes and red dots indicate autolysosomes, n = 4. (D) Quantification of the LC3B fluorescent puncta in (C). (E) After treatment with neomycin, the numbers of live cells in 7 groups (undamaged, neomycin alone, rapamycin-pretreatment, 3-MA-pretreatment, Atg5 siRNA, Becn1 siRNA, and Atg7 siRNA) were determined with the CCK-8 kit, n = 4. For CCK-8 experiments, the values for the normal controls were set to 1. Scale bars: 5 μm. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5.

Autophagy modulates the injury severity in cochlear HCs after neomycin exposure. (A) Immunofluorescence staining with TUNEL and MYO7A in the middle turn of the cochlea after different treatments, n = 3. (B) Immunofluorescence staining for cleaved-CASP3 and MYO7A in the middle turn of the cochlea after different treatments. (C and D) Quantification of the numbers of TUNEL and MYO7A double-positive, as well as cleaved-CASP3 and MYO7A double-positive cells. The numbers and proportions of cleaved CASP3-positive cells and TUNEL-positive cells in the neomycin-treated groups were significantly greater than the undamaged controls. Moreover, the numbers of apoptotic cells were significantly increased by 3-MA and decreased by rapamycin, n = 3. (E) The mRNA levels of 8 apoptosis-related genes were analyzed by qRT-PCR after treatment with neomycin, n = 4. (F) qRT-PCR analysis of the apoptosis-related gene expression in the 3-MA-pretreatment group and rapamycin-pretreatment group after neomycin injury, n = 4. For qRT-PCR experiments, the values for the normal controls were set to 1. Scale bars: 20 μm. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6.

Autophagy affects cell death and apoptosis in HEI-OC-1 cells after neomycin exposure. (A) Apoptosis analysis by flow cytometry after different treatments. The lower left quadrants, lower right quadrants, and upper right quadrants of the images represent live cells, early apoptotic cells, and late apoptotic cells, respectively, n = 4. (B) Quantification of the flow cytometry data. The proportions of dead cells and early apoptotic cells after neomycin treatment were significantly increased compared with the undamaged groups. In addition, the dead and apoptotic proportions could be increased by 3-MA and ATG5 knockdown, and the apoptotic proportions could be reduced by rapamycin. (C) TUNEL and DAPI double staining showing the apoptotic HEI-OC-1 cells after different treatments, n = 4. (D) Cleaved-CASP3 and DAPI double staining confirmed the apoptotic cells after different treatments, n = 4. (E and F) Quantification of the numbers of TUNEL and DAPI double-positive, as well as cleaved-CASP3 and DAPI double-positive cells in (C and D), respectively.

Figure 7.

Autophagy affects the levels of apoptosis-related genes and proteins in HEI-OC-1 cells after neomycin exposure. (A) Western blots with anti-cleaved-CASP3 antibody revealed that the amount of cleaved-CASP3 is significantly increased after neomycin treatment. In addition, the amount could be increased by ATG knockdown and could be reduced by rapamycin, n = 4. (B) Quantification of the western blot in (A). (C) Western blots with anti-cleaved-PARP1 antibody, n = 4. (D) Quantification of the western blot in (C). (E) The mRNA levels of proapoptotic genes and antiapoptotic genes were analyzed by qRT-PCR after treatment with neomycin, n = 3. (F) qRT-PCR analysis of the apoptosis-related gene expression in the ATG5 knockdown group, 3-MA-pretreatment group, and rapamycin-pretreatment group after neomycin injury, n = 3. For qRT-PCR experiments, the values for the normal controls were set to 1. Scale bars: 20 μm. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 8.

Autophagy attenuates oxidative stress in cochlear HCs after neomycin injury. (A) Immunofluorescence staining with Mito-SOX and anti-MYO7A antibodies in the middle turn of the cochlea after different treatments, n = 4. (B) Quantification of the numbers and proportions of Mito-SOX and MYO7A double-positive cells in (A). The numbers and proportions of Mito-SOX-positive cells in the neomycin-treated groups were significantly greater than the undamaged controls. Moreover, the neomycin-induced oxidative stress was significantly increased by 3-MA and reduced by rapamycin, n = 4. (C) The mRNA levels of genes related to redox reactions were analyzed by qRT-PCR after neomycin treatment. The results showed that neomycin downregulated the expression of 5 genes (Sod1, Gsr, Glrx, Nqo1, and Tmx3), n = 3. (D) qRT-PCR analysis of the redox-related gene expression in the 3-MA-pretreatment group and rapamycin-pretreatment group after neomycin injury, n = 3. For qRT-PCR experiments, the values for the normal controls were set to 1. Scale bars: 20 μm. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 9.

Autophagy modulates oxidative stress in HEI-OC-1 cells after neomycin injury. (A) Five different groups of HEI-OC-1 cells were labeled using the Mito-SOX staining kit, n = 4. (B) Flow cytometry data confirmed the results in (A), n = 4. (C) Quantification of the results of flow cytometry in (B). The ROS levels were significantly increased after neomycin treatment compared with the undamaged groups. In addition, the autophagy inducer rapamycin significantly reduced the ROS levels and both the autophagy inhibitor 3-MA and knockdown of ATG5 significantly increased the ROS levels. (D) The mRNA levels of 6 genes related to redox reactions were analyzed by qRT-PCR after neomycin treatment, n = 3. (E) qRT-PCR analysis of the redox-related gene expression in the ATG5 knockdown group, 3-MA-pretreatment group, and rapamycin-pretreatment group after neomycin injury, n = 3. For qRT-PCR experiments, the values for the normal controls were set to 1. Scale bars: 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 10.

Antioxidant treatment promotes the survival of cochlear HCs after neomycin injury. (A) Immunofluorescence staining with Mito-SOX and MYO7A in the middle turn of the cochlea after different treatments, n = 4. (B) Quantification of the numbers of Mito-SOX and MYO7A double-positive cells in (A). (C) Quantification of the MYO7A-positive cells in (A). (D) Immunofluorescence staining for cleaved-CASP3 and MYO7A in the middle turn of the cochlea after different treatments, n = 4. (E) Immunofluorescence staining for TUNEL and MYO7A in the middle turn of the cochlea after different treatments, n = 4. (F and G) Quantification of the numbers of positive cells in (Dand E). The neomycin-induced oxidative stress and apoptosis in HCs were significantly reduced after pretreatment with NAC. Scale bars: 20 μm. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 11.

Antioxidant treatment rescues the cell death and apoptosis in HEI-OC-1 cells after neomycin injury. (A) Six different groups of HEI-OC-1 cells were labeled with the Mito-SOX staining kit, n = 4. (B) Flow cytometry data confirmed the results in A, n = 4. (C) Quantification of the results of flow cytometry in B. The ROS levels were significantly decreased after pretreatment with NAC. (D) Analysis of apoptotic HEI-OC-1 cells by flow cytometry after pretreatment with NAC, n = 3. (E) Quantification of the flow cytometry data. The proportions of dead and apoptotic cells in the ATG5 knockdown groups and 3-MA-pretreatment groups significantly decreased when the increased ROS level was blocked by NAC.

Figure 12.

Antioxidant treatment promotes the survival of HEI-OC-1 cells after neomycin injury. (A) TUNEL and DAPI double staining and (B) cleaved-CASP3 and DAPI double staining showed the number of apoptotic HEI-OC-1 cells after the different treatments, n = 4. (C) Quantification of the numbers of TUNEL and DAPI double-positive, as well as cleaved-CASP3 and DAPI double-positive cells in (A and B), respectively. The apoptotic cells were significantly decreased after pretreatment with NAC. For flow cytometry quantification experiments, the values for the normal controls were set to 1. For all experiments, *P < 0.05, **P < 0.01, ***P < 0.001.