Study On The Biological Function Of Mitochondrial Protein Tim8a

Research Background and purposeDeafness-Dystonia-Optic Neuronopathy(DDON)Syndrome,an X-linked recessive genetic disorder,is characterized by progressively sensorineural hearing impairment,dystonia and optic atrophy.The disease-causing gene is DDP1(deafness dystonia peptide 1)/TIMM8A(translocase of inner mitochondrial membrane 8A),which encodes the DDP1 protein.DDP1,a highly conserved mitochondrial protein homologous to yeast Tim8,is located in the mitochondrial intermembrane space(IMS).DDP1 partners with Tim 13 to form a DDP1-Tim13 complex,which may assist the translocase of mitochondrial membrane protein precursors such as the Tim23.It is known that the substrates of the DDP1-Tim13 complex include citrin and aralal,both of which are involved in the aspartate-malate NADH shuttle pathway.In addition,alterations in expression levels of DDP1 affect mitochondrial morphology.However,the biological function of DDP1 hasn’t been fully understood,since the existing researches were conducted in human cells in vitro,without any knockout animal models of DDP1 gene.In our previous study,we developed a Timm8al mutant mouse line carrying I23fs49X mutation to observe the behavioral changes,and we found the mutant mice recapitulated many phenotypes of DDON syndrome patients.To further clarify the biological function of DDP1,we conducted our study in mutant mice to research the effect of Timm8a1 gene mutation on mitochondrial morphology and mitochondrial functions in vivo,and verify whether the cellular sublocalization of different variants of DDP1 has changed in vitro.Research Methods1.Western Blot was used to test the expression of the Tim8a protein;2.To observe the pathological changes of different tissues by hematoxylin-eosin(HE)staining;3.Electron microscopic analysis of brain samples and quadriceps femoris from the mice to observed the morphologies of mitochondrial;4.Used Western Blot to detect whether the transportation of mitochondrial respiratory chain complexes and mitochondrial proteins that are usually tested have been changed in the Timm8a1123fs49x/y mutant mice;5.Enzymatic staining of muscle biopsy to observe changes in mitochondrial respiratory chain enzyme activities;6.Transfection of plasmids carrying different mutations of DDP1 into HEK293 cells to observe whether the cellular sublocalization of DDP1 has changed;7.Statistical analysisIn this study,all data were analyzed using SPSS20.0.Independent sample t-test was used for comparison between the two groups,P<0.05 means there are significant differences between the two groups.Research Results1.The mutant mice make no detectable Tim8a protein in many tissues;2.The morphologies of different tissues in the mutant mice were normal;3.Abnormal mitochondrial ultrastructures were observed in several brain areas and quadriceps femoris of the mutant mice.Area of the primary auditory cortex has the most significant changes;4.Timm8al gene mutation does not affect the transportation of the mitochondrial proteins Tim23,Tom40 and SOD-2;5.Tim8a deletion does not affect the biogenesis of the complex subunits of the mitochondrial respiratory chain and the enzyme activities of the quadriceps femoris;6.The C66W mutant DDP1 located in the mitochondria;while the truncated DDP1 were diffusely distributed throughout the cell.Conclusion1.The loss of function of Tim8a protein led to abnormal mitochondrial ultrastructures.Frameshift mutation of Timm8a1 gene could neither affect the transportation of the mitochondrial proteins Tim23,Tom40 and SOD-2,nor does it change the biogenesis and the enzyme activities of the respiratory chain.2.The C66W mutant DDP1 was efficiently imported into the intermembrane space of mitochondria,while the truncated DDP1 could not be transferred to the appropriate sublocalization in the mitochondria,indicating that the pathogenic mechanisms of the two different mutations may be different.