Spatio-temporal pattern of c-Jun N-terminal kinase isoforms in the cochleae of C57BL/6J mice with presbycusis

Mitogen-activated protein kinase (MAPK) cascade is a signaling pathway stimulated by many environmental stressors, and it widely participates in cellular physiological and pathological processes. P38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated protein kinase (ERK1/2) are the three classical MAPK pathways. As a MAPK subfamily, JNKs are known to participate in neurodegeneration and senescence (Kumar et al., 2015; Castro-Torres et al., 2019); however, their role in age-related hearing loss remains to be explored. Three genes encode JNKs in mammals, namely, mitogen-activated protein kinase 8 (Mapk8) (JNK1), Mapk9 (JNK2), and Mapk10 (JNK3), which are the main bases for similar sequences and structures of all three paralogs (Castro-Torres et al., 2019; Zeke et al., 2016). JNK1 and JNK2 are widely distributed in different tissues and play roles in insulin resistance, obesity, and inflammation. The distribution of JNK3 is mainly confined to central neurons (Kumar et al., 2015). JNK3 can also be found in the heart, testicles (Davis, 2000), bone marrow (Barreto et al., 2017), and cochlear spiral ganglion neurons (SGNs) (Atkinson et al., 2012).

According to the findings of Nguyen (Nguyen et al., 2015) and Wolfe (Wolfe, 2001), JNKs contain several well-conserved structural features among MAPKs: the CMGC (cyclin-dependent kinases (CDKs), mitogen-activated protein kinases (MAPKs), glycogen synthase kinases (GSKs), and CDK-like kinases (CLKs)) insert protrudes from the C-terminal kinase domain, the short common docking (CD) helix, and the C-terminal helix binds back to the N-terminal domain. JNK isoforms have similar but not identical structures. There are two variable regions for JNK1/2 and three for JNK3, resulting in four isoforms for JNK1 and JNK2 and eight possible isoforms for JNK3, though only three JNK3 isoforms have been characterized (Zeke et al., 2016). A distinct characteristic that distinguishes JNK3 from JNK1/2 is the N-terminal extension.

The structures of JNK1, JNK2, and JNK3 contribute to the similarities and differences in their functions. More specifically, isoforms of both JNK1 and JNK2 actively contribute to c-Jun phosphorylation, but JNK1 appears to be more active than JNK2 as a kinase (Zeke et al., 2016). Palmitoylation is a posttranslational modification. An isoform of JNK3, JNK3α2(L), is palmitoylated at its C-terminus by the DHHC family palmitoyltransferase ZDHHC15, which regulates the subcellular localization and activity of JNK3α2(L), and further regulates axonal development and branching. In contrast, JNK1α2, which has almost the same C-terminal as JNK3α2, cannot be palmitoylated (Yang et al., 2012). Yang et al. suggested that palmitoylation is sensitive to changes in protein sequence and structure; the small difference in COOH terminal amino acids between JNK1 and JNK3, or different protein structures, may be the reason for the unique palmitoylation state (Yang et al., 2012).

Despite differences in the structure and function of JNK isoforms, the activation of all JNK isoforms plays a vital role in promoting neuronal death (Akamine et al., 2020). The enhancement of JNK signaling is prominent in the reactions of astroglia, microglia, and oligodendroglia to anoxia, ischemia, excitotoxicity, and inflammation (Raivich, 2008). In contrast, inhibition of JNK signaling provides substantial and constant neural protection against neurodegeneration (Akamine et al., 2020). Therefore, JNK signaling may be a potential target for the prevention of neurodegenerative diseases (Maroney et al., 1998). Maroney et al. (1998) used the JNK inhibitor CEP-1347 to preserve neurons undergoing apoptosis and found that JNK1 was the major inhibited isoform, revealing that inhibiting the JNK signaling cascade can promote motoneuron survival. Among the studies on single JNK isoforms, JNK3 showed outstanding performance in treating neurological diseases. Notably, the knockdown of JNK3 can downregulate the pro-apoptotic protein Bim, prevent the synthesis of Fas, reduce the release of cytochrome C, decrease phosphorylation of the downstream effector c-Jun, and offer excellent protection in a hypoxic-ischemic brain injury model (Kuan et al., 2003). JNK3 inhibitors may constitute potential therapeutic agents for neonatal brain injury (Pirianov et al., 2007) and acute stroke (Kuan et al., 2003). In the field of hearing loss, studies have shown that inhibiting total JNKs had protective effects against noise or ototoxic drugs that induced hearing loss (Alam et al., 2007; Eshraghi et al., 2007). However, these studies did not explore which JNK isoform plays a more critical role in the process of hearing loss, and the distribution of JNK1/2/3 in the cochleae remains unclear.

Several clinical trials have been conducted to analyze the ability of JNK inhibitors to protect against hearing loss. D-JNKI-1 is an effective inhibitor of the three JNK isoforms, also named AM-111. AM-111 is a minimal conserved domain with a 20 amino acid sequence derived from JNK-interacting protein 1, which interacts with JNK via the JNK-binding domain and inhibits the activation of JNK (Bonny et al., 2001). A clinical phase I/II trial was conducted with AM-111 in 11 patients with acute acoustic trauma exposed to firecrackers (Suckfuell et al., 2007). AM-111 was intratympanic injected at 0.4 mg/mL (n = 7) or 2.0 mg/mL (n = 4). The hearing thresholds at 4 and 6 kHz were improved by 11 dB on days 3 and 30 after treatment from the baseline of 36 dB (Suckfuell et al., 2007). Despite the small sample size and lack of a control group, the trial shows the potential to protect against noise-induced hearing loss in humans. A prospective, randomized, double-blind, placebo-controlled phase II study was completed in 2013 with 210 patients with acute sensorineural hearing loss following acute acoustic trauma within 48 h (Suckfuell et al., 2014). Patients have been treated with 0.4 or 2 mg/mL AM-111 (n = 140) or placebo (n = 70). The outcome showed a significant therapeutic effect of 0.4 mg/mL AM-111 in treating severe-to-profound acute hearing loss but not mild or moderate acute hearing loss, which may result from the high rates of spontaneous recovery (Suckfuell et al., 2014). The following phase Ⅲ study designed to confirm the efficacy of 0.4 mg/mL AM-111 in the recovery of severe to profound idiopathic sudden sensorineural hearing loss (ISSNHL) (Staecker et al., 2019) demonstrated that a single intratympanic dose of 0.4 mg/mL AM-111 provides effective oto-protection against profound idiopathic sudden sensorineural hearing loss (ISSNHL). As these clinical trials showed, the JNK inhibitor of all three isoforms, known as AM-111/D-JNKI-1, worked only when acute hearing loss was severe to profound. How would the expression of JNK1/2/3 change during acute acoustic trauma, and whether their changing tendency is similar or opposite? Despite the acoustic-trauma-induced hearing loss, the JNK pathway plays a role in presbycusis or drug-induced hearing loss and whether different isoforms have particular functions. To date, few studies have provided answers to this question.

To make an effort to provide evidence that JNK isoforms play different roles in hearing loss, we investigated the spatio-temporal pattern of the three isoforms of JNK—JNK1, JNK2, and JNK3—in the cochleae and explored which type of JNK isoform may play a more critical role in age-related hearing loss.

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