The Study On How The Nrf2Influence The Expression Of ATF4in HSOD1^G93A Transgenic Mice

Objective:Amyotrophic lateral sclerosis(ALS) is one of the mostcommon type of motor neuron degeneration.The disease is characterized byupper and lower motor neuron degeneration, upper motor neuron involvementinduced spasticity,dysarthria and dysphagia etc.;lower motor neuroninvolvement as muscle atrophy, fasciculations etc..Nearly90%of ALS casesare sporadic ALS; nearly10%is familial ALS. Among this the G93A (glycineto alanine substitution at the ninety-third codon) is the most commonmutant.SOD1G93Atransgenic mice showed to motor neuron degenerationaggravating,and similar to human motor neuron disease.So the mutantSOD1G93Amice are recognized by the international to the animal model.The experimental studies of hmSOD1transgenic mice that transfecthuman mutant SOD1(human mutant SOD1,hmSOD1),found many waysdamage to motor neurons, for example: oxidative stress, the accumulation ofmis-folded proteins, impaired mitochondrial function, glutamate excitotoxicityetc..In recent years,many scholars have put forward the endoplasmic reticulumstress theory.The endoplasmic reticulum is a multifunctional organelles, ER is a placethat protein synthesis,processing,folding,calcium storage et al..When thecalcium ion balance disorder and incorrectly folded/unfolded proteinaccumulation in endoplasmic reticulum cavity can induce endoplasmicreticulum stress,then triggers the unfolded protein response to cope withendoplasmic reticulum stress.Due to cell protection function,but the cells inplace excessive ER stress can cause cell dysfunction, and cell apoptosis.Appropriate ERS plays important role in protecting cells, however, excessiveERS cause cell-disfunction,leading to apoptosis ultimately.There are threekinds of transmembrane proteins on endoplasmic reticulum surface, respectively PERK (PKR-like ER kinase),ATF6(activating transcriptionfactor6) and IRE1(inositol-requiring enzyme1).There transmembraneproteins are activated when dissociated with GRP78,and then activate therepathways.PERK pathway is activated earliest in three pathway,PERK was activatedby its dimerization and phosphorylation after separation with GRP78,eIF2αwas phosphorylation by pPERK,so that the protein synthesis is slow down orstop.ATF4was activated by peIF2α increase the expression of CHOP,ERADand GADD34,GADD34negative regulation of peIF2α.Nrf2(Nuclear FactorErythroid2-Related Factor2) dissociated with keap1through pPERK,so that itcan into the nuclear and upregulate the expression of phase II enzymes underthe effect of ATF4,which promotes cell survival.Taras Afonyushkin demonstrated that eIF2α upregulated the ATF4translation,while Nrf2up regulation of ATF4transcription.Nrf2and ATF4combined with each other to form dimers,the dimers combined with AREsequences of phase II enzymes gene and then regulate the expression of phaseII enzymes.Thereby regulating cellular redox balance and maintenance ofintracellular homeostasis.This study was developed using the internationally recognized SOD1G93Amice crossbreeding with Nrf2-/-mice obtain experimental mice(Nrf2-/-1SOD1G93Amice)as the research object.Immunohistochemistry andlaser confocal micro-scope were used to research the effect of motor neuronschange in number and shape in different period and the he expression of ATF4site changes.Western blot was used to investigate ATF4changes in differentperiods and effect of Nrf2on the expression of the ATF4.Methods:1Breeding and evaluation of transgenic miceAll mice were kept in SPF (specific pathogen free)environment.Humidity,constant temperature (25-27℃), sterile environment, bred with aseptic waterand particles mice diet.Animal experiments comply with provisions of Hebeiprovince laboratory animal management. All animals were purchased from Jackson laboratory(USA). We knocked down the Nrf2gene from SOD1G93Amice by crossbreeding with Nrf2-/-mice with SOD1G93Amice.The offspringmice were recognized by genotyping for Nrf2and hmSOD1G93Awith tissuefrom tail.2Western BlottingThree groups of Tg mice at pre-symptomatic stage (0point,60days),on-set stage (2point,90days) and end-stage (5point,120days) inaccordance with the standard Snow RJ and their age-matched controllittermates were used.Each spinal cord was dissected for Western blottinganalyses and preserved in-80℃before used.Extracted the protein,determination of ATF4expression in Nrf2+/+G93A transgenic mice ofdifferent periods and expression in Nrf2-/-G93A and Nrf2+/+G93A byWestern bolt method.3ImmunohistochemistryThree groups of Tg mice at pre-symptomatic stage (0point,60days),on-set stage (2point,90days) and end-stage (5point,120days) inaccordance with the standard Snow RJ and their age-matched controllittermates were used.Paraformaldehyde fixed with spinal cord of mice.Oscillation after slicing, stored in4℃before used. The spinal cord of micewere ATF4immunostained with ATF4.4Confocal microscopy4%Paraformaldehyde fixed with spinal cord of mice.Sliced into25um.Similar with the immunohistochemistry procedures,resistingincubation,incubated with FITC(Goat anti rabbit) and Cy5(Goat anti mice)2ndantibody,with nuclear Hochest fluorescence dye incubation.Slides werecovered with antifade solution and then analyzed by confocalmicroscopy(Olympus FV1000).Result: Western blotting method was used to observe the change of ATF4in transgenic mice, it was found that in the control group mice the expressionis less,and no significant differences between the60-120days along with theage growth.But in the lumbar spinal cord of Nrf2+/+G93A mice,ATF4 expression was increased in the pre-symptomatic stage (P>0.05),and it wassignificantly increased in the on-set stage (P<0.05),but it was declined in theend stage (P<0.05).ATF4was significantly increased in the Nrf2+/+G93Athan Nrf2-/-G93A end-stage mice in the end-stage(P<0.05).Immunohistochemistry results showed vacuolization and the numberof motor neuron cell reduced with the progress of the disease.The expressionof ATF4was increased in the pre-symptoms than the control group but it hasno significant difference, there are significantly increased in on-set stage,but inthe end-stage it showed decreased expression. Laser confocal microscopyshowed no significant difference between the expression of ATF4in mice inNrf2+/+G93A and Nrf2-/-G93A in the end-stage.conclusion:ERS can be induced in the SOD1G93Amice,the exprexion ofATF4was increased with the progress of disease;Nrf2can inhibit ATF4expression to a certain extent,aggravating illness progress.