The Role Of TERT Mitochondrial Translocation In Supporting Cells For Postnatal Cochlear Structural Remodeling

Human auditory perception is highly sensitive and accurate,and the complex structure and complete neural circuit of cochlea are the basis of its function.In mammals,the cochlea is immature early in life and needs to undergo a series of structural remodeling to mature and establish complete auditory pathways.During this period,acoustically induced neural activity cannot be generated due to the inability to perceive external sounds.Still,the researchers detected explosive electrical activity in the auditory pathway,which is crucial for the early maturation of the cochlea and the establishment of the auditory pathway.Early spontaneous electrical activity has been shown to originate from K（?）lliker supporting cells that spontaneously release adenosine triphosphate（ATP）through semi-channels and activate purine signaling systems that promote neighboring inner hair cells（IHCs）depolarize and release the neurotransmitter glutamate,which further excites spiral neurons to produce nerve impulse conduction upward.However,what are the starting factors of ATP release from K（?）lliker supporting cells and which molecular mechanisms regulate the dynamic process of ATP release have not been clear.As ATP is synthesized mainly in mitochondria,the mitochondria are considered to be the main sites of spontaneous electrical activity.Telomerase reverse transcriptase（TERT）is the rate-limiting enzyme of telomerase,which is the key component of regulating telomerase activity.It has a telomere dependent effect and regulates the proliferation of cells.Therefore,it is mainly located in proliferative cells,such as embryonic cells,tumor cells and adult stem cells.However,in rodents,TERT can still be expressed in some tissue cells after birth and plays a certain role.In addition to telomere dependent effects,TERT also has non-telomere dependent effects.The main reason is that TERT have nuclear output signals,which can dynamically shuttle between different sub-organelles.Under normal conditions,TERT are mainly located in the nucleus and only a small part of them are located in mitochondria.However,under various factors including DNA damage,oxidative stress or drugs,TERT can be translocated to mitochondria in large numbers to protect mitochondrial function,reduce ROS production and protect mt DNA to reduce cell apoptosis.In addition,overexpression of TERT was found to increase mitochondrial membrane potential（MMP）and[Ca²⁺]c storage capacity.In conclusion,TERT can shift mitochondria and protect mitochondrial function under various injuries and pathological stimuli,playing an important role.Does translocation of TERT to mitochondria do anything other than protect mitochondria from damage?Studies have found that TERT can regulate the survival and maturation of hippocampal neurons through non-telomeric pathways.Although the exact mechanism is not clear,it provides evidence for the involvement of non-telomeric TERT in the regulation of tissue development.Does TERT regulate auditory system through non-telomere dependent effects?If so,what are the mechanisms?By reviewing the literature,there is no report on the role of TERT in the development of the inner ear.Part 1 Expression and subcellular localization of TERT in cochlea of mice in early postnatal periodObjectiveTo analyze the expression and subcellular localization of TERT in mouse cochlea and supporting cells cultured in vitro.MethodPostnatal mice were divided into P1,P4,P7 and P10 groups according to the days after birth.The cochlea of mice was frozen section and immunofluorescence staining was performed to observe the expression and nuclear location of TERT.The cochlear basement membrane of newborn mice（P0-2）was extracted.Sertoli cells were isolated and cultured in vitro.The expression of TERT was observed by immunofluorescence staining and Western Blot detection,and the relationship between TERT and mitochondria was analyzed.The changes of oxidative stress and apoptosis of supporting cells during in vitro culture were analyzed by immunofluorescence staining.Result1.TERT is still expressed in the basic,middle and apical membrane of the early postnatal cochlea from P1 to P10,mainly located in the K（?）lliker supporting cells.The analysis of the distribution of TERT showed that at P1 and P4,TERT were mainly located in the nucleus of supporting cells,partially located in the cytoplasm at P7,and mainly located in the cytoplasm at P10.2.TERT was continuously expressed in cochlear supporting cells in vitro.TERT was mainly located in the nucleus of supporting cells from D1 to D4,and then began to translocate from the nucleus.At D7,TERT was evenly distributed in the cytoplasm and nucleus.At D10,TERT was mainly translocated into the cytoplasm.The colocalization analysis of TERT and mitochondria showed that the TERT in the cytoplasm were well colocalized with mitochondria,and the Pearson correlation coefficients r were 0.63±0.03and 0.71±0.02,respectively.3.There was no significant increase in ROS,Caspase3 and TUNEL levels of cochlear supporting cells in vitro.Conclusion1.TERT is expressed in cochlear support cells in the early postnatal period and gradually migrates to the extracellular mitochondria.2.There was no obvious oxidative stress or apoptosis in cochlear support cells cultured in vitro,and the extracellular mitochondrial translocation of TERT played non-antioxidant and anti-apoptotic effects.Part 2 Mitochondrial function and purine signal detection of cochlear supporting cells cultured during mitochondrial translocation of TERTObjectiveTo analyze mitochondrial function and purine signaling system activation of cochlear supporting cells in vitroMethodThe cochlear basement membrane of newborn mice（P0-2）was extracted and digested,isolated and cultured in vitro from D1 to D10.Changes in MMP and intracellular ATP concentrations were detected to assess changes in mitochondrial function,and extracellular ATP and intracellular Ca²⁺concentrations were detected to assess changes in activation of purine signaling systems.The changes of MMP were analyzed by immunofluorescence.Colorimetric method was used to detect intracellular and extracellular ATP concentration.Intracellular Ca²⁺concentration was analyzed using calcium imaging techniques.Result1.Compared with D1 and D4,MMP of supporting at D7 and D10 cells was increased gradually.The detection of intracellular ATP concentration showed that the intracellular ATP concentration was also at a low level at D1 and D4.However,it increased significantly at D7 and further at D10.2.The results of extracellular ATP concentration detection showed that the extracellular ATP concentration was at a low level at D1 and D4,and significantly increased at D7,and further increased at D10.Calcium imaging results showed that Ca²⁺cell signal intensity was more pronounced in D7 and D10 than in D1 and D4,in addition,the Ca²⁺wave duration was longer in D7 and D10.Conclusion1.ATP synthesis,release and purine signal activation of mitochondria were increased in cochlear supporting cells in vitro.2.The changes in mitochondrial function and purine signaling system are consistent with the time of translocation of TERT to extracellular mitochondria,and TERT may be involved in the activation of purine signaling system.Part 3 Effect of TERT mitochondrial translocation on cochlear structure and functionPurposeTo explore the effect of TERT mitochondrial transfer on cochlear structure and function.MethodThe conditional TERT knockout mice were constructed by CRISPR/Cas9 technique,and were hybridized with Lgr5-Cre ER+/+tool mice.TERTf^{l/fl-Lgr5Cre ER-/+}mice were the experimental group,and TERT^{+/+-Lgr5c Cre ER-/+}mice in the same litter were the control group.TERT gene knockout of cochlear supporting cells was induced by intraperitoneal injection of Tamoxifen.ABR was detected at 14 and 28 days after birth.The changes of cochlear structure were observed by frozen section and immunofluorescence staining.Result1.The results of immunofluorescence staining of cochlear sections showed that TERT was not expressed in supporting cells of the experimental group after intraperitoneal injection of Tamoxifen,while it was still normally expressed in the control group,meaning a successful model.2.Tamoxifen was injected at P4 and P7 to induce knockout,and then ABR was detected at P14 and P28.The results released that the ABR threshold at P14 and P28 in each TERT^{fl/fl-Lgr5Cre ER-/+}group exceed that in the TERT^{+/+-Lgr5c Cre ER-/+}group.The ABR threshold of the experimental groups induced at different times were compared.In P14 auditions,the ABR threshold values of the experimental groups induced at P4 and P7 had little difference;At P28,the ABR threshold（Click,4K-16K Hz）of the experimental group induced by P4exceed that induced by P7,and the difference was statistically significant.It is suggested that the elimination of TERT mitochondrial translocation can affect the function of mouse cochlea.3.After P4 Tamoxifen induction,the cochlea of mice was obtained at P28.Spiral neurons were observed after immunofluorescence staining.The results showed that the number of apical and transiting spiral neurons in the cochlea of the experimental group was reduced compared with that of the control group and the difference was statistically significant,suggesting that the elimination of TERT mitochondrial displacement would affect the survival and maturity of spiral neurons in the cochlea.ConclusionTERT extracellular mitochondrial transfer is involved in regulating the survival and maturation of spiral neurons in early postnatal mice,thus affecting the function of the mouse cochlea.