Elsevier

Neuroscience Letters

Volume 744, 23 January 2021, 135565
Neuroscience Letters

RIP3-mediated necroptosis was essential for spiral ganglion neuron damage

https://doi.org/10.1016/j.neulet.2020.135565Get rights and content

Highlights

  • RIP3 and pRIP3 were upregulated with SGNs injury promoted.

  • RIP3 overexpression promoted while knockdown inhibited ouabain-induced necroptosis.

  • RIP3 knockdown reduced the phosphorylation of MLKL, but not affected MLKL.

Abstract

To identify the role of RIP3 in ouabain-induced necroptosis and offer clinical implications to prevent spiral ganglion neurons (SGNs) from death, ouabain was applied in SGNs derived from fetal rats and injected into Sprague–Dawley rats to construct injury model in vitro and in vivo, respectively. The necroptosis rate of SGNs was determined by flow cytometry and MTT assays. The protein levels and phosphorylation of RIP3 were evaluated using western blotting and immunofluorescence. SGNs injury was observed using H&E staining and immunofluorescence. The hearing function of rats was evaluated by the auditory brainstem response (ABR) and Distortion Product Otoacoustic Emissions (DPOAE) methods. Ouabain caused dose-dependent necroptosis in SGNs and significant loss of SGNs of the cochlear axis in vivo. RIP3 and pRIP3 were upregulated with SGNs injury promoted, and RIP3 overexpression promoted ouabain-induced necroptosis in SGNs in vitro, which could be suppressed by necrostatin-1. RIP3 knockdown inhibited ouabain-induced necroptosis and reduced the phosphorylation of MLKL but no RIP3-dependent effect on the level of MLKL. RIP3 inhibition in vivo protected rats from ouabain-induced hearing damage with reducing ABR threshold shifts and promoting DPOAE amplitudes, while overexpression of RIP3 enhanced ouabain-induced injury that could be partially reversed by necrostatin-1. A decrease of SGNs density and an upregulation of pRIP3 were observed with RIP3 overexpression, which was in contrast when RIP3 was silenced. Therefore, RIP3 was essential for mediating necroptosis in ouabain-induced SGNs damage. Targeting RIP3 may prevent SGNs from death in clinical practice, and finally help the treatment of sensorineural hearing loss.

Introduction

Sensorineural hearing loss (SNHL) is a worldwide common disease that seriously affects individuals' daily life and social communication [1]. It has been estimated by World Health Organization (WHO, 2018, https://www.who.int/en/news-room/fact-sheets/detail/deafness-and-hearing-loss) that 466 million people are confronted with hearing loss in the world caused by various factors including the noise damage from manufacturing, armed forces and urban traffic [2]. The damage to cochlear hair cells and spiral ganglion neurons (SGNs) is one of the principal causes that contribute to SNHL [3]. Besides, our previous study showed the injury of SGNs further hindered the cell replacement therapy, since the degeneration of SGNs provided a more hostile microenvironment for transplantation cells to survive [4]. Thus, identification of the mechanism by which SGNs injury occurred and the exploration key molecules could facilitate to the high efficiency of SGNs replacement and help to protect auditory function.

Necroptosis as a regulated and active cell death pathway, is one of the mechanisms of late ischemic neuronal injuries, and it may become a new pathway for neuroprotection by expanding the therapeutic time window [5]. Our previous study has revealed that necroptosis participated in SGNs injury, since the necroptosis could be prevented by its specific inhibitor necrostatin-1 (Nec-1) [6]. Importantly, receptor interaction proteins 3 (RIP3) was found increased with SGNs injury aggravation, indicating its potential role in the progression of SGNs damage. The activation of RIP3 and induction of RIP1/RIP3 necrosomes were the most well-known mechanism involved in necroptosis [7]. Mixed lineage kinase domain-like (MLKL, a downstream effector of RIP3) activated by RIP3 afterward leads to the rupture of the plasma membrane together with intracellular and organelle membranes, and eventually results in cell death [8]. Thus, we hypothesize RIP3 may be essential for mediating necroptosis in SGNs damage.

The ouabain applied in inner ear is reported to influence SGNs mainly [9]. As a cardiac glycoside, it selectively inhibits the activity of Na+/K+-ATPase and induces complex signaling cascades that lead to the cell apoptosis [10]. Another previous study also demonstrates that ouabain induced damage to both spiral ganglion neurons and cochlear hair cells through p53-dependent pathway [11]. Hence, we employed ouabain to construct the damage model of SGNs in vitro and in vivo. This study aims to identify the role of RIP3 in ouabain-induced necroptosis and offer clinical implications to prevent SGNs from death, and finally to help the treatment of SNHL.

Section snippets

Experimental animals

Female Sprague–Dawley (SD) pregnant rats E15 (200–230 g) of SPF grade were employed for primary SGNs obtainment and male SD rats (200–240 g) were used for in vivo experiments [12]. The male SD rats were treated with 10 mM ouabain in normal saline solution by intraperitoneal injection and injected with normal saline served as the control [13]. Ethic approval could be found in supplementary methods.

Preparation and identification of primary SGNs of rats

The detailed preparation methods of SGNs were presented in supplementary methods. The prepared SGNs

RIP3 and pRIP3 were promoted as ouabain induced SGNs damage increased

The green fluorescence of NSE presented in SGNs in vitro suggested the success of SGN isolation (Fig. 1A). The cell viability was time-dependently decreased with ouabain treatment after SGNs were treated with 500 μM ouabain for 0∼48 h, respectively, and the difference was significant at 24 and 48 h (Fig. 1B). The ouabain at 0∼1000 μM for 24 h induced cell viability reduction in a dose-dependent manner, and ouabain started to dramatically (P < 0.01) reduced cell viability from 500 μM (Fig. 1C).

Discussion

SNHL is generally caused by damages of SGNs and outer hair cells in the cochleae, which contributes to cell death pathways [17]. Generally, programmed necrosis would become one of the alternative ways of cell death when the caspase activity is inhibited and loss the ability in inducing apoptosis [18]. Apoptosis and programmed necrosis might exist simultaneously in cells. Currently, the drug-induced cochlear ototoxicity is very complex. The study of Zheng et al. showed that noise-caused

Credit author statement

Xi Wang and Xiaobo Mao performed the study and interpreted the data, they were major and equal contributors in writing the manuscript, they were listed as co-first authors. Kun Liang and Xiaodong Chen were responsible for data analysis and visualization. Bo Yue and Yang Yang made significant contributions in designing and conceiving the study, and they were major and equal contributors in critically revising the manuscript, they were listed as co-corresponding authors. All authors read and

Funding

This study was supported by National Natural Science Foundation of China (Grant No.: 81700904) and Natural Science Foundation of Hainan Province of China (Grant No.: 817407).

Declaration of Competing Interest

The authors report no declarations of interest.

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