Quantitative profiling of cochlear synaptosomal proteins in cisplatin-induced synaptic dysfunction

Abstract

The disruption of ribbon synapses in the cochlea impairs the transmission of auditory signals from the cochlear sensory receptor cells to the auditory cortex. Although cisplatin-induced loss of ribbon synapses is well-documented, and studies have reported nitration of cochlear proteins after cisplatin treatment, yet the underlying mechanism of cochlear synaptopathy is not fully understood. This study tests the hypothesis that cisplatin treatment alters the abundance of cochlear synaptosomal proteins, and selective targeting of nitrative stress prevents the associated synaptic dysfunction. Auditory brainstem responses of mice treated with cisplatin showed a reduction in amplitude and an increase in latency of wave I, indicating cisplatin-induced synaptic dysfunction. The mass spectrometry analysis of cochlear synaptosomal proteins identified 102 proteins that decreased in abundance and 249 that increased in abundance after cisplatin treatment. Pathway analysis suggested that the dysregulated proteins were involved in calcium binding, calcium ion regulation, synapses, and endocytosis pathways. Inhibition of nitrative stress by co-treatment with MnTBAP, a peroxynitrite scavenger, attenuated cisplatin-induced changes in the abundance of 27 proteins. Furthermore, MnTBAP co-treatment prevented the cisplatin-induced decrease in the amplitude and increase in the latency of wave I. Together, these findings suggest a potential role of oxidative/nitrative stress in cisplatin-induced cochlear synaptic dysfunction.

Keywords: Cisplatin; Cochlear synaptosome; MnTBAP chloride; Nitrative stress; Ototoxicity; Synaptic dysfunction.

Figure 1:. Experimental design.

Baseline ABRs were recorded in 5-week-old male CBA/J mice. At the beginning of 6^th week, the mice were treated with 3 mg/kg of cisplatin daily for 5 days and/or 10 mg/kg of MnTBAP daily for 7 days. On the 8^th day, ABRs were recorded, and synaptic dysfunction was assessed using wave I amplitude and latency. Then, the mice were sacrificed, and synaptosomal proteins were extracted from the cochlea. Changes in the abundance of cochlear synaptic proteins were assessed using mass spectrometry (LC-MS/MS).

Figure 2:. Cochlear synaptosome protein enrichment.

Enrichment of synaptosomal proteins was verified by immunoblotting with anti-CtBP2, a pre-synaptic biomarker in cochlear ribbon synapses. Immunoblot indicated that CtBP2 was detected at much higher levels in the synaptosome fraction than in the total homogenate and supernatant fraction. GAPDH, which was used for normalization, was expressed in all fractions.

Figure 3:. Cisplatin-induced changes in the hearing thresholds as well as the amplitude and latency of wave I.

(A) Cisplatin treated animals showed significantly higher threshold shifts compared to saline at 8, 16, 24 and 32 kHz frequencies (p=0.004, p=0.031, p=0.008 and p=0.006, respectively). (B) Cisplatin-treated mice showed a significant decrease in amplitude for all stimuli (8, 16, 24 and 32 kHz) (p=0.007, p=0.001, p=0.0002 and p=0.0097). (C) Cisplatin-treated mice showed a significant increase in latency of wave I for all frequencies (p=0.03, p=0.001, p=0.0044 and p=0.0013). Both these changes suggest cochlear synaptic dysfunction with cisplatin treatment. Results were expressed as mean ±SD, n=6, (*p<0.05, **p<0.01, ***p<0.001), and saline-treated mice were used as controls.

Figure 4:. Cisplatin-induced changes in cochlear synaptosomal proteins

(A) Flow chart represents the stepwise mass spectrometry data analysis. (B) The volcano plot depicts the cisplatin-induced changes in the abundance of cochlear synaptic proteins. For high and low abundant proteins, a fold change value of ±1.5 was chosen. The data was plotted as log₁₀p-value against log₂ fold-change. The data on the right and left of the vertical red lines above the horizontal red line indicate the proteins whose abundance was significantly altered by cisplatin treatment. This figure was generated using Rstudio. The results are the mean of three biological replicates. (C) Immunoblots indicate that cisplatin treatment induced a decrease in the expression levels of CtBP2 and SNAP25, verifying the results of the MS/MS analysis. The images are representative of three biological replicates.

Figure 5:. Effect of MnTBAP co-treatment on cochlear synaptosomal proteins.

(A) Venn diagram shows the distribution of differentially abundant proteins in all three treatment groups (cisplatin=351 proteins, MnTBAP co-treated=468 proteins, and MnTBAP=363 proteins). (B) Network analysis using a string data base (version 11.5) identified the involvement of three networks. (C) Heatmap illustrates the relative changes in the abundance of proteins after cisplatin, MnTBAP+cisplatin, and MnTBAP alone treated groups. All groups were normalized to the control group, and proteins with fold change ± 1.5 (p<0.05) were included in this analysis. Results are expressed as the mean of 3–4 biological replicates.

Figure 6:. Effect of MnTBAP co-treatment on cisplatin-induced changes in amplitude and latency of wave I

(A) MnTBAP co-treatment significantly attenuated cisplatin-induced reduction in the amplitude of wave I generated with stimuli at 24 and 32 kHz frequencies (p=0.01 and p=0.024) and increase in latency of wave I generated with stimuli at 24 and 32 kHz frequencies (p= 0.005, p=0.013) Results were expressed as mean ± SD, n=6, (*p<0.05, **p<0.01), saline-treated mice were used as controls.