SUMOylation Of Spastin Mediates Microtubule Dynamic In Regulation Of Hippocampal Neuron Growth And Development

Objective:This study is to investigate whether spastin can be modified with SUMOylation(Small ubiquitin-like modifiers)and how SUMOylation of spastin regulates microtubule dynamics and affects neuronal growth and development.Materials and methods:First,the binding of spastin to SUMO in vivo and in vitro was detected by GST pull down,co-IP,immunocytofluorescence staining and BIFC.The potential SUMOylation sites of spastin were predicted by software,and were identified by immunofluorescence chemistry and molecular experiments.Second,the mutant plasmids based on the SUMOylation sites were transfected into the neurons,and we observed the neurite growth by immunofluorescence chemistry.Third,spastin was cotransfected with SUMO1 or SENP1 into COS1 cells to determine the effect of spastin's microtubule severing activity on different SUMOylation states.And,immunofluorescence staining was used to analyze how spastin K427 R affected the dynamics of microtubules.Four,overexpression of spastin K427 R detect that it promoted or inhibited which type of microtubule posttranslational modification and observe the effect of spastin K427 R on the growth of hippocampal neurons.Finally,immunofluorescence staining was used to observe the effect of spastin's microtubule severing activity in different microtubule posttranslational modification states and determine the effect of spastin on the neurite growth of hippocampal neurons.Results:(1)The results of pull down and co-IP experiments showed that spastin could physically interact with SUMO.Immunochemical staining showed that spastin and SUMO were co-located in hippocampal neuron.BIFC assay showed that clear yellowish green fluorescence was observed in the cotransfected with SUMO1-N YFP and spastin-C YFP,and the fluorescence distribution was similar to the shape of microtubules.(2)The predicted spastin had three potential SUMOylation sites.It was found that spastin K427 AAA lost the microtubule severing activity.Furthermore,it was found that spastin 427 A and spastin 427 R lost the activity of microtubule severing.And the co-IP results showed that Spastin K427 R did not bind to SUMO1.(3)Spastin K305 AAA and K530 AAA could significantly promote axonal development,while spastin K427 AAA and panAAA including mutation K427 could not promote axonal development.(4)Compared with the control group,the fluorescence intensity of microtubule in COS1 cells co-transfected by spastin and SUMO was significantly decreased.And the fluorescence intensity of microtubule co-transfected by spastin and SENP1 was increased.(5)The results of GST pull down and co-IP showed that spastin K427 R protein could interact with tubulin,and spastin K427 R was colocated with acetylation and detyrosination of microtubule by immunocytochemical staining.(6)Spastin K427 R can resist nocodazole depolymerization microtubules,and resist repolymerization of nocodazole depolymerized microtubules.(7)Overexpressing spastin K427 R in COS1 cells,the fluorescence intensity of acetylation and detyrosination of microtubule were significantly stronger than the control group.However,the fluorescence intensity of tyrosination of microtubule was not significantly different.The expression of spastin K427 R in HEK293 cells lead to significantly increase acetylation and detyrosination of microtubule,while tyrosinated and total tubulin were not significantly changed.(8)The fluorescence intensity in COS1 cells transfected with spastin and TTLL4,HDAC or TTL6 was significantly lower than that the control group transfected with spastin.And the fluorescence intensity of microtubule in the COS1 cells cotransfected with spastin K427 R and spastin was significantly highr than that in the control group,and the microtubule was cut into fragments of varying lengths.(9)When spastin and TTLL4,TTLL6 or HDAC6 co-overexpressed in neurons,the number of neuronal process branches was significantly increased,but the number of neuronal branches was significantly decreased when it was treated with tubacin.Those spastin co-expressed with spastin K427 R,the number branches was significantly decreased.Conclusions:(1)Spastin can be modified by SUMOylation and the SUMOylation site of spastin is located at the 427 th spermatogenic;the target of Spastin can be modified by SUMOylated spastin was microtubule;(2)The SUMOylation of spastin promoted its microtubule severing activity,whereas deSUMOylation of spastin inhibited microtubule severing activity and it inhibited the growth of neuronal processes.(3)Spastin K427 R can be combined with tubulin,and Spastin K427 R could promote the acetylation and detyrosination of microtubule so that it inhibit microtubule dynamics.Spastin K427 R inhibited the formation of neuronal processes.(4)Detyrosination of microtubule promoted the severing activity of spastin so that it was beneficial to the branching effect in neurons.But Acetylation of microtubule inhibited the severing activity of spastin and formed fewer microtubule fragments,so it inhibited the neuronal branching effect of spastin.