Role Of Dynaminin The Endocytosis Of IHC And Connectionwith Hearing

Hearing loss is the most common disability in developed countries, among which sensorineural hearing loss(SNHL) is the most commondeafness. It is important to study the pathophysiology of SNHL. Acoustic signal forms in inner hair cells(IHCs) of cochlea, which can release excitatory neurotransmitters to the synaptic gap. Neurotransmitters promote postsynaptic membrane to form the action potential. The whole pathway includes a process that mechanical-chemical-electrical signals conversion, and then inducethe central nervous system to form hearing.Continued effective release of neurotransmitter from IHC to the synaptic gap requires an efficient vesiclerecycle. It has been proved that clathrin-mediated endocytosis(CME) may be the main mechanism of vesiclerecycle. Dynamin plays an essential role in CME of retinal cells and nerve cells. Dynamin forms a spiral structure at the neck of clathrin-coated pit, and thenhydrolyze GTP to shrink to cut the neck to release the pit from membrane. The defect of Dynamin will lead to the CME disorder, reduce the number of synaptic vesicle and decrease the neurotransmitter release. Dynamin“knock down” mice exhibit a visual impairment. There is little study for theexpress and function of Dynamin in IHCs, and we don’t know it’s function in hearing development.In this study, we explorethe expression pattern of each sub-type of Dynamin in IHCs,seek the connection between Dynamin and hearing in different age of mice. We use the whole cell patch-clamp to test Dynamin`s loss of function to regulate the endocytosis of IHC. With the entire test, we try to investigate the Dynamin through methods of morphology, molecular biology and physiology both in vivo and vitro, to clear its role in the CME of IHC.Methods:1. The animals were adult Kunming mouse wild type. Took cochlea basilar membrane from the mice; the expression and distribution of each subtype of Dynamin in cochlear basement membrane of adult mouse were distinguished by immunofluorescence, and detected by thelaser confocal microscope, to ensure each type of Dynamin expressed in IHC.2. After the immunofluorescenceimage observation of Dynamin, the auditory brainstem response(ABR) thresholdwas recorded from the Kunming mice on postnatal 3 weeks(young), 10 weeks(adult), and 16 months(aged). Discuss the relation between the expression pattern of Dynamin and the age.3. IHC in cochlear from each age group was examined for the morphology and expression of Dynamin subtypesby immunofluorescence. Discuss the relation between the expression pattern of Dynamin and the age.4. The expression of Dynamin subtypes m RNA in each age group was detected with theq RT-PCR(quantificational real-time polymerase chain reaction), find out the influence that age to Dynamintranscription level, and then to discuss the relevance between Dynamintranscription level and hearing.5. To test the membranecapacitance change of IHC after inhibiting the functionof Dynamin by whole cell patch-clamp techniques, and to reveal Dynamin’s function in endocytosis.Results:1. Three subtypes of Dynamin are expressed in the inner hair cell of mice. In IHC s, Dynamin-1 was expressed in the whole inner hair cell. Dnamin-2 distributed in the cytoplasm. Dynamin-3 highly expressed under nucleus of inner hair cell, near by thespiral ganglion.2. ABR threshold showed no significant difference between the group of young and adult,but threshold of the aged group increased comparing with groups of bothyoung and adult(P<0.05).3. Dynamin-1、Dynamin-2 expressed in the whole inner hair cell in the group of young and adult. In the aged group, Dynamin-1 was lost beneath the nucleus, and Dynamin-2 could only be found near the nucleus. In addition, Dynamin-3 expressed in the basilar region which was close to spiral ganglions in each age group, however, Dynamin-3 in HIC of aged group shown to be less than that of young and adult.4. The q RT-PCR revealed that m RNA of Dynamin-1 reduced with age(P<0.05),m RNA of Dynamin-2 increased to a peak in the adult group and then reduced with age. Meanwhile, m RNA of Dynamin-3 could not be detected.5. Whole cell patch-clamp test shown an increase of Δ Cm in IHC after Dynaminfunction was blocked by Dyngo-4a.Statistical analysis didn’t carry out due to the lack of data.Conclusion: Three subtypes of Dynamin are expressed in cochlear hair cells. The expression tendency of Dynamin-1 and Dynamin-2 differs with ageing. Dynamin plays an essential role in clathrin-mediated endocytosis(CME) of IHC. Different subtypes of Dynamin were involved in CME at different ages. Normal CME requires Dynamin-1 subtype in the young mice and at the adulthood Dynamin-2 may replace Dynamin-1’s role, which suggests that Dynamin-2 in IHC involved in the hearing developing from the young to the adult and maintain normal function in whole lifetime. Dynamin-1 contributes to hearing in young mice. However, we think the role of Dynamin-3 in hair cells is unclear. CME was abnormal after Dynamin was blocked. This study expands a new research orientation for the pathophysiology of SNHL as well as the way of prevention and cure.