Study On Cytotoxicity Induced By Truncated Mutation Of Deafness Gene DFNA5

Part 1: Study on expression distribution and subcellular localization of DFNA5Objective: To investigate the expression and subcellular localization of DFNA5 in rats by detecting the expression of DFNA5 in multiple organs of newborn rats,frozen sections of rat cochlea and immunofluorescence staining of HEI-OC1 cells.Methods: The liver,lung,kidney,spleen,heart,muscle,brain and cochlea were harvested from postnatal day 1 Sprague–Dawley rats.Total protein was extracted and the expression of DFNA5 in various tissues and organs was detected by western blot.The level of DFNA5 in the cochlea and the subcellular localization of DFNA5 in HEI-OC1 cells were observed by immunofluorescence staining.Results: DFNA5 was expressed in cochlea,muscle,brain,spleen,heart,kidney,lung and liver,and the expression of the cochlea was the highest.The results of frozen section showed that DFNA5 was endogenously expressed in hair cells and spiral ganglion neurons in the rat cochlea.Endogenously expressed DFNA5 in subcultured HEI-OC1 cells was evenly distributed in the cytoplasm.Conclusion: The expression of DFNA5 is detected endogenously in multiple tissues and organs of rats.The expression of DFNA5 in the cochlea is mainly expressed in hair cells and spiral ganglion neurons.Endogenous DFNA5 is mainly distributed in cytoplasm.Part 2: Toxic effects of different DFNA5 fragments in mammalian cellsObjective: To explore the mechanism of cytotoxicity induced by DFNA5 truncation mutation in vitro by transfecting different kinds of DFNA5 fragments into HEK-293 T cells and HEI-OC1 cells.Methods: The DFNA5 full-length(DFNA5-FL),amino-terminal fragment(DFNA5-NT),carboxy-terminal fragment(DFNA5-CT)and exon 8 skip deletion fragment(DFNA5-?exon8)recombinant plasmid vector were constructed.The correctly identified recombinant plasmid vectors were transfected into HEK-293 T cells and HEI-OC1 cells.The morphological changes of the cells were observed under laser confocal microscopy 36 h after transfection.The concentration of LDH in the supernatant of the cell culture medium was detected and the percentage of dead cells was calculated.Results:(1)After transfecting the HEK-293 T cells with the recombinant plasmid vector carrying the EGFP tag at the amino terminal of the DFNA5 fragment for 36 h,the cells in the NT and ?exon8 groups was abnormal,showing a slight shrinkage of the cell body,a unsmooth surface of the cell membrane,and an uneven distribution of intracellular fluorescence.There is little difference between the percentage of dead cells in each group,although statistically significant(P<0.05).After transfecting the HEK-293 T cells with the recombinant plasmid vector carrying the Myc tag at the carboxy terminal of the DFNA5 fragment for 36 h,the percentage of cell death in the NT and ?exon8 groups was significantly increased,compared with that in the negative control group,the FL group or the CT group(P<0.05).After transfecting the HEK-293 T cells with the recombinant plasmid vector carrying the Flag tag at the amino terminal of the DFNA5 fragment for 36 h,the percentage of dead cells in the NT group was significantly increased and the percentage of dead cells in the ?exon8 group was slightly increased,both of which were statistically significant compared with that in the negative control group,the FL group or the CT group(P<0.05).(2)After transfecting the HEI-OC1 cells with the recombinant plasmid vector carrying the Myc tag at the carboxy terminal of the DFNA5 fragment for 48 h,LDH assay showed that there was no significant difference in the percentage of dead cells in each group.Conclusion: By constructing recombinant plasmid vectors carrying different DFNA5 fragments and mammalian cells transfection,we confirmed that the DFNA5-NT and the DFNA5-?exon8 can cause abnormal cell morphology and cell death,which was preliminarily proved to be pyroptosis;the cytotoxicity of the mutant DFNA5 was derived from the amino-terminal fragment of the truncated DFNA5,but not the 41 abnormal amino acids that were incorrectly translated for the frameshift mutation.The amino terminal structure is crucial for the pyroptosis-induced function of the truncated DFNA5,and the amino terminal protein modification may affect the function.The DFNA5-FL or the DFNA5-CT did not cause pyroptosis.Part 3: The effect of DFNA5 truncation mutation on mouse inner earObjective: To explore the mechanism of the DFNA5 mutation-induced deafness by transfecting recombinant adeno-associated virus vector carrying human DFNA5 with exon 8 skip deletion mutant(DFNA5-?exon8)into mouse inner era via round window membrane microinjection.Methods: 36 wild-type C57BL/6J mice were randomly divided into four groups.Animals in the experimental group(12 animals),the GFP overexpressing group(12 animals)and the artificial perilymphatic fluid(APF)group(6 animals)were microinjected with 2?L r AAV2/9-DFNA5-?exon8-GFP,r AAV2/9-GFP or APF respectively into scala tympani via round window membrane by postauricular approach while the animals in the control group(6 animals)were kept untreated.All animals were subjected to ABR test 21 d after surgery.Then all animals were sacrificed and the cochleas were harvested for immunofluorescence staining and observation by laser confocal microscopy.Results:(1)The results of ABR test showed that the ABR thresholds in the left ear of animals in the experimental group,GFP overexpression group and APF group were significantly increased compared with that of the control group(P<0.05).There was no significant difference among the ABR thresholds in the left ear of animals in the experimental group,GFP overexpression group and APF group(P>0.05).There was no significant difference among the ABR thresholds in the right ear of animals in each group(P>0.05).(2)In the GFP overexpressing group,the inner hair cells of the basal turn,middle turn and apical turn of the cochlear basement membrane showed green fluorescence expression,and the transfected positive cells showed a decreasing trend.The average transfection efficiency of inner hair cells in the basal turn,middle turn and apical turn was 82.7%,75.0%,44.7%,respectively.The transfected positive cells were arranged neatly and in normal morphology.No cytotoxicity such as swelling,atrophy or shedding was observed.No green fluorescent expression was observed in the outer hair cells.(3)In the transfected positive cells which were mainly inner hair cells in the experimental group,the intracellular green fluorescence distribution was unevenly located in the cytoplasm and in a granulated manner.The transfected positive cells were arranged neatly and in normal morphology.No cytotoxicity such as swelling,atrophy or shedding was observed.Conclusion: The recombinant adeno-associated virus vector carrying the gene of interest can be transfected into the inner ear of mice by round window membrane microinjection.The AAV2/9 is mainly transfected into inner hair cells.The DFNA5-?exon8 leads to an abnormal granulated expression in the inner hair cells of mouse cochlea.But no ototoxicity was observed in the mice after transfection.