Screening And Optimization Of The Neuroprotective Transferrin Mutations For Ischemia Stroke

Cerebral apoplexy,also known as"stroke",is an acute cerebral vascular disease caused by the sudden death of local brain cells due to insufficient blood flow.It includes ischemic stroke and hemorrhagic stroke.The incidence of ischemic stroke is higher than that of hemorrhagic stroke,accounting for about 87%of the total number of stroke.Clinic survey exhibited that stroke has become the first cause of death in China and the leading cause of disability among Chinese adults.Previous studies have found that transferrin has a neuroprotective effect on ischemic stroke by reducing iron overload.Transferrin is an iron-binding plasma glycoprotein that controls the level of free iron in biological fluids.It consists of two separate parts,the N-lobe and the C-lobe,each of which can be individually bound to an iron.The process of transferrin binding to iron is reversible and has a very high affinity for Fe（III）,but gradually decreases as pH drops below neutral,releasing iron at about pH 5.6.Based on the structure characteristics of transferrin,the experiment mutated the transferrin in order to obtain versions with stronger neuroprotective effect.The experiment analyzed possible mechanisms to provide new strategy for stroke treatment.The main results gained are list as follows:Firstly,the cerebral stroke was made in C57BL/6 mice.Three days after stroke,the mice were perfused for brain frozen sections.Using antibodies agaist ferritin,TF,and the markers of neurons and glial cells,MAP2 and GFAP,The experiment found that ferritin and TF significantly increased in the focal region of stroke,and can be co-labeled with neurons and glial cells,indicating the iron overload and TF up-regulation exists in both cells.Secondly,the experiment modified the transferrin to obtain mutants with either smaller molecular weight or lower iron release rate.The amino acid residues that play a key role in the release of iron are the dimeric residues of K296/K206 at the N-lobe and the trimeric residues of R632/K534/D634 at the C-lobe of transferrin.Mutation at Lys296 in N lobe or Asp634 in C lobe to alanine Ala resulted lowest iron release rate at pH5.6.C-lobe Asp634 is also associated with the release of transferrin in a receptor-dependent manner.In addition,since independent N-lobe and C-lobe can bind iron ions alone,The experiment compared the neuroprotective effect of N-lobe,C-lobe,full-length transferrin with or without single point mutation.The experiment have successfully expressed and purified the N-lobe deletion TF（TFc）,C-lobe deletion TF（TFn）,N-lobe deletion and C-lobe site-directed mutation TF（TFcmt,D634A）,C-lobe deletion and N-lobe site-directed mutation TF（TFnmt,K296A）,full-length double-lobe site-directed mutated TF（TFmt,K296A/D634A）,and wild-type full-length TF.Finally,in vitro oxidative stress model and erastin-induced ferroptosis model were applied for evaluating the purified TF and its mutations on their neuroprotective effect on stroke.（1）In N2a cells and A172 pro cell lines,the experiment establish the oxidative stress model by using different concentrations of hydrogen peroxide（H₂O₂）and evaluated the neuroprotective effect of different types of transferrin.The results showed that the neuroprotective effect of TFmt（K296A/D634A）)on both types of cells was significantly higher than that of TF,indicating that the protective function might be weaken to the ability to release iron by K296A/D634A.The neuroprotective effects of TFnmt（K296A）,TFn,TFcmt（D634A）and TFc on the both cell lines were significantly higher than those of TFmt（K296A/D634A）,and the protective effects of TFc were the most significant in all versions of TF proteins,indicating that the C-lobe receptor-dependent function might also contribute to the anti-oxidative stress function of TF.（2）According to the recent reports about erastin-induced ferroptosis,the experiment used50μM erastin to induce the ferroptosis in N2a cells and A172 cells.The experiment observed if TF can protect the erastin-induced ferroptosis.In the N2a cells,TFcmt（D634A）,TFc and Holo-TFcmt（D634A）,Holo-TFc enhanced the erastin-induced ferroptosis.In A172 cells,TFnmt（K296A）,TFn,TFc and Holo-TFnmt（K296A）,Holo-TFn,Holo-TFcmt（D634A）,Holo-TFc enhanced the erastin-induced ferroptosis.This might indicate that the mechanism of the TF’s neuroprotective role on oxidative stress may not have a direct relationship with the erastin-induced ferroptosis.As summary,the experiment screened for the most effective N-lobe deletion version of TF（TFc）for the protection on neurons and glial cells oxidative stress.The experiment also suggested that the neuroprotective effect of transferrin on oxidative stress may be improved by weaken its ability for iron release and might also related with its receptor-dependent function on C-lobe,but not related to the ferroptosis induced by erastin.These information may provide valuble novel directionfor the ischemic stroke neuroprotective mechanism research and potential clinic translation of transferrin.