The Role Of T-Type Calcium Channels In Spiral Ganglion Neurons In Age-Related Hearing Loss

Age-related hearing loss?AHL?is the most common sensory disorder in the elderly population that affects tens of millions of people worldwide.AHL,known as presbycusis,refers to the symmetry and progressive decline of binaural auditory function with the increase of age,and is characterized by decreased in high-frequency hearing sensitivity.Yet,the molecular mechanisms underlying AHL are still unknown.It has been shown that degeneration of spiral ganglion neurons?SGNs?plays a critical role in AHL.SGNs are the primary afferent neurons of the auditory system and provide the vital link between hair cells and the central auditory nuclei.SGNs convey signals from hair cells to the brain in a manner that preserves the amplitude,frequency,and temporal features of sound information.However,the mechanism of SGNs degeneration in AHL need to be further explored.T-type calcium channels,known as low-voltage activated calcium channels?LVA?,are a subfamily of voltage-gated calcium channels,and play an important role in mediating neuronal Ca²⁺homeostasis.Three subtypes of T-type calcium channels Ca_v3.1,Ca_v3.2 and Ca_v3.3 all express in SGNs.Administration of trimethadone or ethuximide,two antiepileptic drugs that may block T-type calcium channels,can delay age-related SGNs loss and preserve ABR thresholds in elderly mice.Our previous study showed that the mRNA levels of Ca_v3.1,Ca_v3.2 and Ca_v3.3 in SGNs of AHL mice were significantly increased.These studies suggest that T-type calcium channels in SGNs are involved in AHL,however,whether and how alterations of T-type calcium channels in SGNs contribute to AHL are still unknown.To investigate the mechanism of T-type calcium channels-induced SGNs degeneration contribute towards AHL,we measured the expression of T-type calcium channels in SGNs of old mice?a reported AHL animal model?as well as detectd the effect of T-type calcium channels on SGNs apoptosis.Our findings may provide valuable insights into the mechanism underlying AHL.Objective:To investigate the role of T-type calcium channels on SGNs in AHL.Methods:1)Evaluation of auditory function of C57BL/6 mice at different ages by measuring auditory brainstem response?ABR?;2)Real-time PCR and immunofluorescence staining were used to examine the mRNA and protein expression level of three subtypes of T-type calcium channels in SGNs from young and old mice;whole-cell patch clamp technique was used to record T-type calcium currents in SGNs from young and old mice.3)SGNs morphometry was examined by hematoxylin and eosin staining.The SGNs density in cochlea tissue sections from young and old mice was examined by performed histological analysis;Expression of apoptosis-related protein such as AIF,cleaved caspase-3,calpain1 and calpain2 in SGNs from old mice were detected by immunofluorescence,Real-time PCR,and Western-Blot;4)T-type calcium channels inhibitors and calpain2 inhibitors were respectively incubated on SGNs to observe the nuclear translocation of AIF.Results:1.ABR hearing thresholds from old mice were significantly elevated than those of young mice at low frequencies and exceeded the upper limits of ABR system at the middle and high frequencies;2.We examined the expression of Ca_v3.1,Ca_v3.2 and Ca_v3.3 in SGNs from young and old mice.We found all of them were present in young and old SGNs.Ca_v3.1,Ca_v3.2 and Ca_v3.3 were increased significantly in SGNs from old mice at the mRNA expression level.Ca_v3.1 and Ca_v3.2 were increased significantly in SGNs from old mice at the protein expression level.T-type calcium currents significantly increased in SGNs from old mice compared with young ones.3.We found that the cochlea from young mice displayed no loss of SGNs,while in old mice,they displayed severe loss of SGNs.The SGNs densities were decreased significantly in old mice,compared with young mice;4.Our data showed that AIF was located mainly in the cytoplasm of SGNs in young mice.In old mice,there was increased translocation of AIF from cytoplasm to nucleus.The calpain 2 was increased significantly in SGNs from old mice at mRNA and protein level.By contrast,calpain1 expression were statistically unchanged between young and old mice;5.Application of 300 mM D-galactose could induce the translocation of AIF from cytoplasm to nucleus in SGNs.Incubation of T-type calcium channels blocker Z944 and calpain2 inhibitor ALLM may inhibit the AIF translocation.Conclusion:In summary,we found T-type calcium channels were up-regulated in SGNs from old mice.AIF translocation to the nucleus in SGNs of old mice,associated with loss of SGNs,could be activated by T-type calcium channels through activation of calpain2.Taken together,our results demonstrate that T-type calcium channels may induce SGNs apoptosis which may contribute to AHL.