The Effect Of Dynamin-related Protein 1(DRP1) On Neuron Outgrowth And Development And Its Mechanism

Dynamin related protein 1(DRP1),which belongs to the motility protein family of large GTPases,mediates mitochondrial division.More than two decades of research have produced a lot of information about DRP1-mediated mitochondrial division;however,in higher eukaryotes The majority of DRP1 is in the entire cytoplasm,while a small part of the protein DRP1 is found to be assembled on the outer mitochondrial membrane.The biological function of most of DRP1 distributed in the cytoplasm is still unknown.This paper mainly discusses from two aspects.First,it is clear whether the length and complexity of neuronal processes will change after DRP1 protein overexpression,knockdown and phosphorylation;second,it is clear that the cell morphology changes after DRP1 is knocked out As well as the expression of microtubule-related proteins,explore the mechanism that affects the changes in neuronal processes.In order to clarify whether the length and number of neuronal processes will change after the overexpression,knockdown and phosphorylation of DRP1 protein,and whether the morphology of neurons will change,we used wild-type rat fetal rat cerebral cortex at the embryonic stage of 18 days.Isolate primary neurons as a model for primary culture in vitro.The neurons were electrotransfected with an electroporator,and the medium was changed for 24 hours.It was found that compared with the control plasmid,the neurons of the overexpressed DRP1-WT plasmid grew slightly faster.After 4 days of culture in vitro(DIV4),immunofluorescence staining was performed with specific antibodies,and the total length and number of neuronal processes were statistically analyzed using ImageJ software.The results showed that(1)the length of neuronal processes overexpressing DRP1-WT plasmid Significant growth,the number of neuron processes increased significantly;(2)After overexpression of DRP1-K38A(DRP1 dominant negative mutation)plasmid and DRP1-KD plasmid,the length of neuron processes was significantly shortened,and the number of neuron processes was significantly reduced.This suggests that the DRP1 protein may affect the morphological changes of neurons;(3)Overexpression of DRP1-S616D and DRP1-S637A plasmids significantly increase the length of neuronal processes and the number of neuronal processes;(4)Overexpression of DRP1-S616A and DRP1-After the S637D plasmid,the length of neuron processes was significantly shortened,and the number of neuron processes was significantly reduced.This suggests that phosphorylation of DRP1 protein may also affect the morphological changes of neurons.In the early stage of the laboratory,it was found that there is a certain interaction between DRP1 protein and cytoskeleton tubulin.In order to verify the role of cytoskeleton tubulin in cells,we constructed a DRP1 knockout cell line in human embryonic kidney cells 293 cells.Verification experiment.Subsequently,we transfected DRP1-WT,DRP1-S616D,DRP1-S637A,DRP1-S616A and DRP1-S637D plasmids in the DRP1-KO cell line by liposome transfection.The results showed that(1)the expression of acetylated tubulin was significantly reduced after DRP1 was knocked out;(2)the expression of acetylated tubulin increased after overexpression of DRP1-S616D and DRP1-S637A plasmids;(3)After overexpression of DRP1-S616A and DRP1-S637D plasmids,the expression of acetylated tubulin was significantly reduced;(4)Exogenous and endogenous co-immunoprecipitation experiments showed that DRP1 protein and cytoskeleton tubulin had a certain interaction Effect;(5)DRP1 protein inhibits the assembly of microtubules to a certain extent.It is suggested that DRP1 protein and phosphorylation modification may affect the acetylation modification of tubulin.Subsequently,we used immunofluorescence experiments to detect the intracellular localization and morphological changes of tubulin,acetylated tubulin.The results of statistical analysis found that after DRP1 was knocked out,acetylated tubulin appeared to be fragmented,with scattered positioning and reduced expression.It shows that DRP1 protein may affect the acetylation level of tubulin to some extent and inhibit the assembly of microtubules.