Aging of the auditory system is associated with the incremental production of reactive oxygen species (ROS) and the accumulation of oxidative damage in macromolecules, which contributes to cellular ...malfunction, compromises cell viability, and, ultimately, leads to functional decline. Cellular detoxification relies in part on the production of NADPH, which is an important cofactor for major cellular antioxidant systems. NADPH is produced principally by the housekeeping enzyme glucose‐6‐phosphate dehydrogenase (G6PD), which catalyzes the rate‐limiting step in the pentose phosphate pathway. We show here that G6PD transgenic mice (G6PD‐Tg), which show enhanced constitutive G6PD activity and NADPH production along life, have lower auditory thresholds than wild‐type mice during aging, together with preserved inner hair cell (IHC) and outer hair cell (OHC), OHC innervation, and a conserved number of synapses per IHC. Gene expression of antioxidant enzymes was higher in 3‐month‐old G6PD‐Tg mice than in wild‐type counterparts, whereas the levels of pro‐apoptotic proteins were lower. Consequently, nitration of proteins, mitochondrial damage, and TUNEL+ apoptotic cells were all lower in 9‐month‐old G6PD‐Tg than in wild‐type counterparts. Unexpectedly, G6PD overexpression triggered low‐grade inflammation that was effectively resolved in young mice, as shown by the absence of cochlear cellular damage and macrophage infiltration. Our results lead us to propose that NADPH overproduction from an early stage is an efficient mechanism to maintain the balance between the production of ROS and cellular detoxification power along aging and thus prevents hearing loss progression.
G6PD overexpression contributes to overcome cochlear oxidative damage, prevents hair cell and neuronal loss, and, thereby, delays age‐related hearing loss. Congenital levels of NADPH are increased, thus inducing the expression of antioxidant genes via Nrf2, enhancing antioxidant defense systems, reducing oxidative‐derived damage accumulation, and preventing mitochondrial dysfunction and apoptosis of irreplaceable cochlear cell populations in adult mice.
Mitogen-activated protein kinases (MAPK) such as p38 and the c-Jun N-terminal kinases (JNKs) are activated during the cellular response to stress signals. Their activity is regulated by the ...MAPK-phosphatase 1 (DUSP1), a key component of the anti-inflammatory response. Stress kinases are well-described elements of the response to otic injury and the otoprotective potential of JNK inhibitors is being tested in clinical trials. By contrast, there are no studies exploring the role of DUSP1 in hearing and hearing loss. Here we show that
expression is age-regulated in the mouse cochlea.
gene knock-out caused premature progressive hearing loss, as confirmed by auditory evoked responses in
mice. Hearing loss correlated with cell death in hair cells, degeneration of spiral neurons and increased macrophage infiltration.
mouse cochleae showed imbalanced redox status and dysregulated expression of cytokines. These data suggest that DUSP1 is essential for cochlear homeostasis in the response to stress during ageing.
Insulin-like growth factor 1 (IGF-1) deficiency is an ultrarare syndromic human sensorineural deafness. Accordingly, IGF-1 is essential for the postnatal maturation of the cochlea and the correct ...wiring of hearing in mice. Less severe decreases in human IGF-1 levels have been associated with other hearing loss rare genetic syndromes, as well as with age-related hearing loss (ARHL). However, the underlying mechanisms linking IGF-1 haploinsufficiency with auditory pathology and ARHL have not been studied. Igf1-heterozygous mice express less Igf1 transcription and have 40% lower IGF-1 serum levels than wild-type mice. Along with ageing, IGF-1 levels decreased concomitantly with the increased expression of inflammatory cytokines, Tgfb1 and Il1b, but there was no associated hearing loss. However, noise exposure of these mice caused increased injury to sensory hair cells and irreversible hearing loss. Concomitantly, there was a significant alteration in the expression ratio of pro- and anti-inflammatory cytokines in Igf1+/− mice. Unbalanced inflammation led to the activation of the stress kinase JNK and the failure to activate AKT. Our data show that IGF-1 haploinsufficiency causes a chronic subclinical proinflammatory age-associated state and, consequently, greater susceptibility to stressors. This work provides the molecular bases to further understand hearing disorders linked to IGF-1 deficiency.
Stress-activated protein kinases (SAPK) are associated with sensorineural hearing loss (SNHL) of multiple etiologies. Their activity is tightly regulated by dual-specificity phosphatase 1 (DUSP1), ...whose loss of function leads to sustained SAPK activation.
gene knockout in mice accelerates SNHL progression and triggers inflammation, redox imbalance and hair cell (HC) death. To better understand the link between inflammation and redox imbalance, we analyzed the cochlear transcriptome in
mice. RNA sequencing analysis (GSE176114) indicated that
cochleae can be defined by a distinct profile of key cellular expression programs, including genes of the inflammatory response and glutathione (GSH) metabolism. To dissociate the two components, we treated
mice with N-acetylcysteine, and hearing was followed-up longitudinally by auditory brainstem response recordings. A combination of immunofluorescence, Western blotting, enzymatic activity, GSH levels measurements and RT-qPCR techniques were used. N-acetylcysteine treatment delayed the onset of SNHL and mitigated cochlear damage, with fewer TUNEL
HC and lower numbers of spiral ganglion neurons with p-H2AX foci. N-acetylcysteine not only improved the redox balance in
mice but also inhibited cytokine production and reduced macrophage recruitment. Our data point to a critical role for DUSP1 in controlling the cross-talk between oxidative stress and inflammation.
In a Dutch consanguineous family with recessively inherited nonsyndromic hearing impairment (HI), homozygosity mapping combined with whole-exome sequencing revealed a MPZL2 homozygous truncating ...variant, c.72del (p.Ile24Metfs∗22). By screening a cohort of phenotype-matched subjects and a cohort of HI subjects in whom WES had been performed previously, we identified two additional families with biallelic truncating variants of MPZL2. Affected individuals demonstrated symmetric, progressive, mild to moderate sensorineural HI. Onset of HI was in the first decade, and high-frequency hearing was more severely affected. There was no vestibular involvement. MPZL2 encodes myelin protein zero-like 2, an adhesion molecule that mediates epithelial cell-cell interactions in several (developing) tissues. Involvement of MPZL2 in hearing was confirmed by audiometric evaluation of Mpzl2-mutant mice. These displayed early-onset progressive sensorineural HI that was more pronounced in the high frequencies. Histological analysis of adult mutant mice demonstrated an altered organization of outer hair cells and supporting cells and degeneration of the organ of Corti. In addition, we observed mild degeneration of spiral ganglion neurons, and this degeneration was most pronounced at the cochlear base. Although MPZL2 is known to function in cell adhesion in several tissues, no phenotypes other than HI were found to be associated with MPZL2 defects. This indicates that MPZL2 has a unique function in the inner ear. The present study suggests that deleterious variants of Mplz2/MPZL2 affect adhesion of the inner-ear epithelium and result in loss of structural integrity of the organ of Corti and progressive degeneration of hair cells, supporting cells, and spiral ganglion neurons.
ABSTRACTBetaine‐homocysteine S‐methyltransferases (BHMTs) are methionine cycle enzymes that remethylate homocysteine; hence, their malfunction leads to hyperhomocysteinemia. Epidemiologic and ...experimental studies have revealed a correlation between hyperhomocysteinemia and hearing loss. Here, we have studied the expression of methionine cycle genes in the mouse cochlea and the impact of knocking out the Bhmt gene in the auditory receptor. We evaluated age‐related changes in mouse hearing by recording auditory brainstem responses before and following exposure to noise. Also, we measured cochlear cytoarchitecture, gene expression by RNA‐arrays and quantitative RT‐PCR, and metabolite levels in liver and plasma by HPLC. Our results indicate that there is an age‐dependent strain‐specific expression of methionine cycle genes in the mouse cochlea and a further regulation during the response to noise damage. Loss of Bhmt did not cause an evident impact in the hearing acuity of young mice, but it produced higher threshold shifts and poorer recovery following noise challenge. Hearing loss was associated with increased cochlear injury, outer hair cell loss, altered expression of cochlear methionine cycle genes, and hyperhomocysteinemia. Our results suggest that BHMT plays a central role in the homeostasis of cochlear methionine metabolism and that Bhmt2 up‐regulation could carry out a compensatory role in cochlear protection against noise injury in the absence of BHMT.—Partearroyo, T., Murillo‐Cuesta, S., Vallecillo, N., Bermúdez‐Muñoz, J. M., Rodríguez‐de la Rosa, L., Mandruzzato, G., Celaya, A. M., Zeisel, S. H., Pajares, M. A., Varela‐Moreiras, G., Varela‐Nieto, I. Betaine‐homocysteine S‐methyltransferase deficiency causes increased susceptibility to noise‐induced hearing loss associated with plasma hyperhomocysteinemia. FASEB J. 33, 5942–5956 (2019). www.fasebj.org
We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. The first procedure consists of perforation of ...the tympanic bulla, termed bullostomy; the second one is a transtympanic injection. Both emulate human clinical intratympanic procedures. Chitosan-glycerophosphate (CGP) and Ringer´s Lactate buffer (RL) were used as biocompatible vehicles for local drug delivery. CGP is a nontoxic biodegradable polymer widely used in pharmaceutical applications. It is a viscous liquid at RT but it congeals to a semi solid phase at body temperature. RL is an isotonic solution used for intravenous administrations in humans. A small volume of this vehicle is precisely placed on the Round Window (RW) niche by means of a bullostomy. A transtympanic injection fills the middle ear and allows less control but broader access to the inner ear. The safety profiles of both techniques were studied and compared by using functional and morphological tests. Hearing was evaluated by registering the Auditory Brainstem Response (ABR) before and several times after microsurgery. The cytoarchitecture and preservation level of cochlear structures were studied by conventional histological techniques in paraformaldehyde-fixed and decalcified cochlear samples. In parallel, unfixed cochlear samples were taken and immediately frozen to analyze gene expression profiles of inflammatory markers by quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR). Both procedures are suitable as drug delivery methods into the mouse middle ear, although transtympanic injection proved to be less invasive compared to bullostomy.
Age-related hearing loss (ARHL) is the most common sensory deficit in the elderly. The disease has a multifactorial etiology with both environmental and genetic factors involved being largely ...unknown. SLC7A8/SLC3A2 heterodimer is a neutral amino acid exchanger. Here, we demonstrated that SLC7A8 is expressed in the mouse inner ear and that its ablation resulted in ARHL, due to the damage of different cochlear structures. These findings make SLC7A8 transporter a strong candidate for ARHL in humans. Thus, a screening of a cohort of ARHL patients and controls was carried out revealing several variants in
, whose role was further investigated by in vitro functional studies. Significant decreases in SLC7A8 transport activity was detected for patient's variants (p.Val302Ile, p.Arg418His, p.Thr402Met and p.Val460Glu) further supporting a causative role for SLC7A8 in ARHL. Moreover, our preliminary data suggest that a relevant proportion of ARHL cases could be explained by SLC7A8 mutations.
Unfortunately, cisplatin has multiple adverse effects and causes irreversible hearing loss (HL) in a large proportion of patients, which severely impacts their quality of life.1 Here, we confirm that ...treatment with LPT99, a small molecule inhibitor of the apoptosome, preserves hearing levels in a rat model of cisplatin-induced HL and prevents apoptosis in cisplatin-exposed HEI-OC1 cells. ...more effective agents are urgently needed, preferably formulated for local (intratympanic) administration to avoid interfering with the antineoplastic activity of cisplatin. LPT99-co-treated cochleae also showed less dilatation of strial microvasculature, particularly in the middle turn and exhibited higher levels of Kir4.1, a marker of intermediate cells (Figure 4I and O). Because the stria vascularis is the entry point of cisplatin into the cochlea and a long-term accumulation site, it has emerged as an important target to prevent cisplatin ototoxicity.2 Further studies are needed to demonstrate a direct effect of LPT99 on strial cells.
Excessive exposure to noise damages the principal cochlear structures leading to hearing impairment. Inflammatory and immune responses are central mechanisms in cochlear defensive response to noise ...but, if unregulated, they contribute to inner ear damage and hearing loss. Transforming growth factor β (TGF-β) is a key regulator of both responses and high levels of this factor have been associated with cochlear injury in hearing loss animal models. To evaluate the potential of targeting TGF-β as a therapeutic strategy for preventing or ameliorating noise-induced hearing loss (NIHL), we studied the auditory function, cochlear morphology, gene expression and oxidative stress markers in mice exposed to noise and treated with TGF-β1 peptidic inhibitors P17 and P144, just before or immediately after noise insult. Our results indicate that systemic administration of both peptides significantly improved both the evolution of hearing thresholds and the degenerative changes induced by noise-exposure in lateral wall structures. Moreover, treatments ameliorated the inflammatory state and redox balance. These therapeutic effects were dose-dependent and more effective if the TGF-β1 inhibitors were administered prior to inducing the injury. In conclusion, inhibition of TGF-β1 actions with antagonistic peptides represents a new, promising therapeutic strategy for the prevention and repair of noise-induced cochlear damage.
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