| Age related hearing loss(ARHL)is the most common chronic sensory disorder in the elderly.Elderly people over 70 years of age are prone to ARHL.According to WHO(WHO)estimates,in 2025,the population over the age of 60 will reach 1 billion 200 million,of which 500 million people are expected to suffer from age related hearing loss.ARHL is caused by a variety of factors,including the aging of the cochlea,environmental,genetic predisposition(genetic predisposition,disease)and health disorders.The main pathological features of ARHL include loss of hair cells,stria atrophy,loss of spiral ganglion neurons,and changes in the central auditory pathway.Not surprisingly,ARHL is a complex,multifactorial disorder,involving environmental,genetic,health and nutritional factors.Human heritability studies suggest that 25-75%of the variance in ARHL has a genetic basis.This common age-related disorder is closely related to oxidative stress.Search to date suggests that oxidative stress and mitochondrial DNA deletion(mtDNA)play a major role in pathophysiology of ARHL.Finding of numerous studies implicate that oxidative damage in the cochlea reflects an age-related decline in the antioxidant defenses and/or an age related increase in ROS levels which plays a crucial role in the development of ARHL.It has been proposed that targeting of oxidative stress by pharmacological or genetic interventions can help to prevent or slow ARHL.Most studies have been reported that supplementation with certain antioxidants can prevent/slow ARHL.Nevertheless,the effect of antioxidants supplementation in the prevention of ARHL is presently controversial and inconclusive.Besides,the short duration of action of these chemical inhibitors or anti-oxidants limits their clinical utility in the treatment of ARHL.Therefore,the development for antioxidant therapy in ARHL requires an understanding of oxidative stress mechanisms underlying the ARHL that may overcome this obstacle by opening doors to supply new treatment options.Mammalian target of rapamycin(mTOR)is a serine/threonine kinase that coordinates numerous cellular processes.It assembles into two multiprotein complexes:the rapamycin sensitive mTOR complex 1(mTORC1)and rapamycin-insensive mTORC2.Many aging and age-related diseases have been shown to be related to mTORC1 signaling.Over the last decade,a handful of studies have estimated that inhibition of mTORC1 pathway confers protections against a growing list of age-related pathologies.Accumulating evidence suggested that mTORC1 acts downstream of Caloric restriction(CR)and CR reduces mTORCl activity in some mammalian tissues.On the one hand,mTORC1 signaling is thought to play a part in mediating longevity and health benefits as a result of CR.On the other hand,CR is also known to extend life span in yeast,worms,fruit flies,spiders,birds,and monkeys and delays the progression of a variety of age-associated diseases such as cancer,diabetes,cataract,and age-related hearing loss(ARHL)in mammals.Thus,all these studies of the complex relationship of mTORC1 with age-associated diseases suggesting a potential role for mTORC1 in ARHL.But so far,the exact role of the mTORC1 signaling pathway in age-related hearing loss is unclearIn the current study,we first found a significantly increase of mTORC1 activity in aging NSE of cochlea.Inhibition of mTORC1 with Rapamycin or NSE-specific deletion of Raptor can attenuate the age-related hearing loss in C57BL/6J mice.Furthermore,at the mechanistic level,using NSE-specific conditional TSC1 knockout mouse model,we showed that sustained mTORCl activation can break the redox balance in NSE,thus causing early-onset death of cochlear hair cells and progressive hearing loss.Here in this paper,we are the first to clarify the critieal role of mTORC1 pathway in ARHL using these two unprecedented useful animal models.These results indicate that targeting the mTORC1 pathway may shed new light on preventing and treating ARHL.The scientific questions that should be solvedThis thesis needs to be discussed around the following scientific problems:1)The relationship between mTORC1 signaling pathway and age-related hearing loss.2)The specific mechanism of mTORC1 signaling pathway in the regulation of age-related hearing loss.3)The role of peroxisomes in age-related hearing loss.4)How autophagy plays a role in the regulation of mTORC1 signaling pathways in the development of age-related hearing loss.Research methods and experimental results obtainedThe C57BL/6J mouse strain is the most widely used animal model for the study of ARHL because it carries the recessive early-onset hearing loss-susceptibility allele(Cdh23753A).The ABRs,an electrophysiological hearing test that detects evoked potentials in the auditory pathway from the cochlea to the upper brainstem,was used to monitor the progression of ARHL in C57BL/6J mice.We confirmed that the mean ABR hearing thresholds were significantly elevated at all frequencies(4 kHz,8 kHz,16 kHz,24 kHz,and 32 kHz)in 12-month-old C57BL/6J mice compared with those in 2-month-old C57BL/6J mice,indicating that the older mice(12-month-old)displayed age-related hearing loss.HC and spiral ganglion cell(SGC)degeneration are hallmarks of ARHL.Consistent with the ABR results,basal regions of the cochleae exhibited HC and SGC losses in 12-month-old WT mice.To investigate whether mTORC1 signaling is involved in the development of ARHL,we first examined the levels of S6 phosphorylation at 235/236(P-S6),a downstream target of mTORC1 that is frequently used as an in vivo indicator of mTORC1 activity in the cochlear hair cells of C57BL/6J mice.Iumunolabeling for P-S6 in middle turn OHCs was significantly increased in 12-month-old mice compared with that in 2-month-old mice.Western blot analysis using sensory epithelium tissues also demonstrated increased P-S6(235/236)and P-P70S6K.In contrast,P-Akt(Ser473)remained unchanged with age,suggesting specific mTORC1 activation in NSE with unchanged mTORC2 signaling.To evaluate the location of the increased P-S6(235/236)in key regions of the cochlea,SGNs and the organ of Corti,P-S6(235/236)was immunolabeled in cochlear paraffin-embedded sections.In 2-month-old mice,P-S6 was rarely observed in hair cells(IHCs and OHCs)and Deiter cells,but strong expression was observed in the outer and inner pillar cells.P-S6 expression levels were significantly upregulated in hair cells and Deiter cells in aged mice;however,no obvious changes were detected in either the outer or inner pillar cells or the SGNs.Collectively,these results demonstrated that mTORCl activity in NSE increased with age in mice,raising the possibility that dysregulated mTORC1 signaling plays a role in the development of ARHL.Rapamycin is proved to be a very effective inhibitor of the mTORC1 signaling pathway.We first studied the effect of reducing mTORC1 signaling pathway on hearing in C57BL/6J mice by intraperitoneal injection of rapamycin.We found that when the C57BL/6J mice three months from the beginning of the continuous injection of a Rapamycin,ABR results showed that Rapamycin of mice did not produce side effects,sixth months when the injection group and the control group began hearing level difference:injected mice than in the control group common hearing threshold of hearing threshold than the control group the low,with the increase of the age of the difference between the two groups becomes more obvious.This showed that the control group showed signs of age-related deafness with age,but the mice injected with Rapamycin were resistant to age related hearing loss.The results of Western blot showed that the expression of P-S6 in the cochlear tissue of mice was greatly reduced after injection of Rapamycin,which suggested that Rapamycin could prevent the occurrence of senile deafness by decreasing the level of mTORC1 signaling pathway.Whole-body rapamycin treatment affects not only the NSE but also other cochlear cell types,such as SGNs and the stria vascularis.Therefore,although inhibition of mTORC1 signaling by rapamycin alleviates ARHL,it is not clear whether inhibition of mTORC1 signaling in NSE alone is sufficient to prevent ARHL.To answer this question,we generated mice exhibiting conditional ablation of the mTORC1-specific component Raptor in the NSE using a Cre recombinase expression cassette.The morphology of Raptor-/-mice was similar to that of their control littermates,indicating no markedly obvious developmental defects.Based on H&E staining of cochlear sections,Raptor-/-mice also exhibited no visible structural alterations in the inner ear compared with WT mice.Next,to detect whether inactivation of the mTORC1 signaling pathway prevents ARHL,we carried out electrophysiological tests of cochlear function at different ages.At 3 months,the mean ABR thresholds in Raptor-/-mice were virtually identical to the controls.There was a significant elevation in ABR thresholds characteristic of ARHL at 9,12,and 17 months of age in the controls.By 17 months,the mean thresholds had increased by>40 dB compared to those seen at 3 months of age.However,in Raptor-/-mice,there were no significant increases in ABR thresholds characteristic of ARHL at 9,12,or 17 months of age.The mean ABR threshold shifts were significantly smaller at all frequencies tested.DPOAE was used to detect a low-level sound generated by the active mechanism of OHCs emitted to the ear canal.At 3 months,similar DPOAEs were observed between Raptor-/-mice and controls.In contrast,at later stages,the DPOAEs in Raptor-/-mice were significantly lower than those in control mice.However,the differences were smaller than those seen in corresponding frequency regions via ABRs.This result indicates that differences in neuronal origin(e.g.,reduction in synaptic ribbons)exert additional effects on the ABR responses.In agreement with the hearing test results,control mice showed significant hair cell loss at the age of 17 months;most of the OHCs were absent,whereas only minor loss of OHCs was observed in the Raptor-/-mice.Quantification confirmed a greater retention of hair cell numbers in Raptor-/-mice.Thus,inactivation of mTORCl signaling promoted hair cell survival in the aging inner ear.Furthermore,we investigated whether the number of synaptic ribbons,which were immunostained with antibodies to CtBP2/Ribeye,was different between aged controls and Raptor-/-mice.Although most IHCs remained intact in control mice,numbers of synaptic ribbons in the remaining IHCs were strikingly reduced compared with those in Raptor-/-mice.We also counted SGNs in the middle region and observed no significant differences between Raptor-/-and control mice,supporting the involvement of synapses rather than cell bodies in the deterioration of hearing in aged eontrol mice.Taken together,these results provide evidence that inactivation of mTORC1 signaling in NSE alleviates ARHL.Since mTORC1 activity is significantly increased in aged cochlea,and inactivation of mTORC1 signaling in NSE alleviates ARHL,we used a genetic approach to increase mTORC1 signaling selectively in NSE to further determine the mechanism through which NSE mTORC1 activity promotes ARHL.mTORC1 is under tonic inhibition by the TSC1/Tsc2 complex,and TSC1 deletion results in constitutively increased mTORC1 signaling.We crossed TSC1flox/flox mice with Atohl-Cre transgenic mice to produce TSC1flox/flox;Atohl-Cre mice.We found that there was no abnormality in the auditory system development of TSC1-/-mice,but a decrease in hearing thresholds occurred from P30 days,accompanied by loss of hair cells.Over time,auditory hair loss was more severe 4 months of age TSC1-/-mice were almost completely lost.These results suggest that overexpression of the mTORC1 signaling pathway has no effect on the formation of mouse hearing,but does have a significant effect on the maintenance of hearing.Premature loss of hair cells leading to progressive deafness in TSC1/-mice.mTORC1 affects many downstream metabolic signaling pathways,including Notch,Erk,Wnt,oxidative stress,protein synthesis,and so on.There is a close relationship between age related hearing loss and oxidative stress,and the imbalance of redox reaction is the main factor leading to the development of age-related hearing loss.In view of this,we focus our research on oxidative stress.Our results showed that the expression of marker 4-HNE and 3-NT of oxidative stress was significantly increased in TSC1/-mice.This suggests that oxidative stress plays an important role in hearing maintenance in TSC1-/-mice.In order to verify this conclusion,we change the level of reaction was detected by Q-PCR to maintain the redox balance of the enzyme,the results showed that the expression level of mRNA gene was knocked out in the preoxidation of TSCI increased significantly after addition of antioxidant genes,and the expression level of mRNA decreased significantly.Subsequently,we treated TSC1-/-mice with antioxidant NAC,and data showed that NAC treatment could protect the cochlea from oxidative stress in mice.From this we can infer that oxidative stress is probably the main factor leading to hair cell death and hearing loss in TSC1-/-mice.The two organelles of mitochondria and peroxisomes are the main sites of intracellular oxidative stress.Mitochondria are the major organelles responsible for oxidative stress and the major site of energy metabolism in cells.In recent years,many reports have reported that age-related deafness is related to mitochondrial genes.We observed by transmission electron microscopy that in P30,though the hearing loss in mice,the mitochondria were still in good shape:the bilayer membrane was normal and the cristae of mitochondria were very obvious.This suggests that mitochondria may not have obvious problems at this time,and oxidative stress is responsible for TSC1-/-mice,and deafness is caused by other factors.In addition,peroxisome dysfunction may lead to accumulation of ROS and is associated with hearing loss.Peroxisome is a very dynamic organelle derived from the endoplasmic reticulum,whose primary cellular function is to interact with other cellular components and participate in the regulation of the redox state of cells.Transmission electron microscopy showed that the hair cells of TSC1-/-mice appeared peroxisome morphology defects:some peroxisomes appeared multiple crystalline nucleus,many peroxisomal membrane structure burst.These results suggest that the destruction of peroxisomes in mTORC1 over activated mice is the most important link in hair cell oxidative stress,leading to hair cell death and loss of hearing in mice.How does TSC-mTORCI over activation affect peroxisomes in mice?We found that the TSC1/TSC2 complex was localized to the peroxisomes of hair cells.This result suggests that the TSC-mTORC1 signaling pathway may play a role in peroxisomes.According to numerous biochemical and genetic studies,mTORC1 negatively regulates autophagy.Recent studies have also revealed that autophagy plays essential roles in the mouse inner ear,and autophagy attenuates noise-induced hearing loss by reducing oxidative stress.As rapamycin is an effective autophagy activator and protects TSC1-/-cochlear hair cells from injury,we wondered whether autophagic activity was affected in the cochleae of TSC1-/-mice.Modification of microtubule-associated protein 1 light-chain 3(LC3)expression levels,specifically the shift from LC3I to LC3II expression,is one of the initiating steps in autophagosome formation.Our results showed that rapamycin injection induced the up-regulation of LC3? in TSC1-/-mice cochleae.Our data also revealed decreased levels of P62/SQSTM1,a specific substrate that binds to LC3 to facilitate the degradation of ubiquitinated proteins.Therefore,we speculated that autophagic fl ux dysfunction is one cause of the early-onset degeneration of hair cells in TSC1-/-mice.Recent reports have shown that rats injected i.p.with aminoglycoside antibiotics induce autophagy.However,the basal levels of autophagy were very low in the cochleae because little LC3II was detected.To increase the sensitivity of detection,we injected neomycin(an aminoglycoside antibiotic)into TSC1-/-/GFP-LC3 mice and control mice once per day for 5 consecutive days starting at P30 to increase autophagic flux.The autophagic response in auditory hair cells within the organ of Corti was then analyzed using immunofluorescence.Immunofluorescence analysis of the basilar membrane revealed that punctuate GFP fluorescence in OHCs in TSC1-/-/GFP-LC3 mice was much higher than that in littermate controls.Additionally,we used TEM to examine ultrastructural autophagic processes in auditory hair cells within the organ of Corti,and our data revealed that several autophagosomes(round,double-membraned structures)appeared 5 days after neomycin injection in GFP-LC3 mice,whereas no autophagosomes were observed in TSC1-/-/GFP-LC3 mice.Together,these observations indicate that autophagy is dysregulated in TSC-/-mice.To sum up,TSC-mTORC1 signaling pathway plays an important role in the process of hearing loss.Targeting the mTORC1 pathway may prevent and treat age related hearing loss.Research significance:?.We first elucidated the TSC/mTORC1 signaling pathway play a very important role in presbycusis;through down-regulation of mTORC1 signaling pathway can be used for anti senile deafness,which provides a therapeutic target and a good theoretical basis for the prevention and treatment of age related hearing loss.?.The important role of peroxisomes in ear hair cells is elucidated.The destruction of peroxisomes may also cause damage to the auditory hair cells,leading to deafness.(?).The TSC/mTORC1 signaling pathway regulates the process of deafness by modulating the balance of intracellular redox.The study of auditory function in the TSC/mTORC1 signaling pathway will help to deepen our understanding of age-related deafness. |
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