Changes Of XBP1and ATF6MRNA Expression In Spinal Cord Of Familial Amyotrophic Lateral Sclerosis Model HSOD1^G93A Transgeneic Mouse

Amyotrophic lateral sclerosis (ALS) is a kind of neurologicaldegenerative disease. It belongs to motor neuron disease, both upper andlower motor neurons are involved. Before the disease, patients often appearsome nonspecific symptoms. For example: body fatigue, poor-sportsendurance and so on. The limitation about the activities in unilateral orbilateral distal end of limbs and movement weekness appear as the onsetsymptom. The muscles of proximal end of the limbs, trunk and neck areweekness, paralysis and atrophy, finally the patient ofen die in respiratoryfailure or lung infection caused by respiratory muscles atrophy and weekness.The incidence of ALS is1to2per100,000, person. Approximately10%ofALS patients are inherited (fALS), and most cases of ALS are sporadic(sALS). There is most common characteristic of motor neuron disease inamyotrophic lateral sclerosis, unknown etiology, poor curative effect andunfavourable prognosis, the pathogenesis of ALS involves a variety of factors.Several studies about fALS show that a variety of gene mutation residedin fALS, zinc-dependent superoxide dismutase1(SOD1) gene, TDP-43gene,FUS/TLS gene and so on. The greatest contribution to ALS come from thediscovery of mutations in the SOD1, ALS1is a subtype of ALS with SOD1Mutations.That can account for20%of familial ALS. SOD1gene locates onchromosome21, with five exon and four introns. More than100SOD1genemutations about ALS have been reported. These mutations existed widely inall the five exons. Mostly mutations are missense and deletion. Therefor,human SOD1gene was transduced into mice successfully. This hSOD1(hSOD1) mutations transgenic mice used widely.There was a lot of theory about the pathogenesis of ALS, for example: genetic mutation, oxidative stress and so on. Recently，several studies haveshown that endoplasmic reticulum stress play an important role in ALS.Endoplasmic reticulum is an organelle in the cytoplasm of all mammals. It is acomplicate, meshy closed piping system, undertaked important physiologicalfunction in cells. Endoplasmic reticulum involved into the pathologicalprocess of nucleolus and mitochondria. The correlation of endoplasmicreticulum stress and ALS has been confirmed both in experimentation onanimals and ALS patients.Normally, endoplasmic reticulum was the monitor of the quality of mostprotein and acted as an important intracellular calcium store. Whenendogenous and extrinsic stress factors encroached on cell, the processing ofprotein is disturbed and a large number of unfolded protein was produced.Then, over-accumulated unfold proteins resulted in unfolded protein response(UPR). The abnormal protein was cleared by the way of ER associateddegradation (ERAD). Appropriate ERS play important role in protecting cells,however, excessive ERS cause Cell-dysfunction, leading to neuron apoptosisultimately.There are three kinds of transmembrane protein in Endoplasmicreticulum.they are IRE1(inositol-requiring enzyme1)、ATF6(activatingtranscription factor6) and PERK (PKR-like ER kinase), GRP78/BIP andthere transmembrane protein are dissociated with the activation of therepathway. The activation of the IRE1can splice XBP1mRNA, encode splicedXBP1(sXBP1) protein. The activation of the ATF6was transferred into Golgibody and cut to activited p50-ATF6, sXBP1and p50ATF6could translocatesto the nucleus, Combined with ER stress response element (ERSE) andincrease the expression of some ERS related protein. PERK phosphorylateseukaryotic initiation factor2α (eIF2α), which results in the decreasedtranslation of lots of protein to protecting cell from damage.This hSOD1mutation transgenic mouse is most widely usedexperimental model. Endoplasmic reticulum stress existed in the hSOD1G93Amice. This study is to find the change of the mRNA of XBP1, sXBP1and p50 ATF6in the hSOD1G93Amice to find the relation between ERS and ALS.Methods: Part one: hSOD1G93Atransgenic mice and identifyHemizygous males B6SJL-Tg (SOD1-G93A)1Gur/J and femalesB6SJLF1/J+/+breeding, Obtaining progeny mice, shearing tip of its tail,extracting DNA, PCR amplification, and the PCR product waselectrophoresed on agarose gel analysis. After identifed, we can obtainhSOD1G93Atransgenic mice, and wtSOD1mice without hSOD1.Part two:To research the change of mRNA about XBP1、sXBP1and p50ATF6in the spinal cells of hSOD1G93Atransgenic mice.(1)Experimental group：28days old hSOD1G93Atransgenic mice,35daysold hSOD1G93Atransgenic mice,49days old hSOD1G93Atransgenic mice,63days old hSOD1G93Atransgenic mice, symptoms of hSOD1G93Atransgenic mice3mice (about90days), end-stage hSOD1G93Atransgenicmice (about120days). Every group chooses wtSOD1mice from same broodas control.(2)Drawn materials: at each points, anesthetized mouse with10percent chloral hydrate, remove the waist pulp of mouse, liquid nitrogenfrozen swiftly. Then specimens was transfered into-80℃.(3)Real-time RT-PCR: extract the total RNA with Trizol reagent,measured the concentration of total RNA, reversed transcription to cDNA,real time RT-PCR With cDNA templates using SYBRGreen, observed eachindicator in each group.Results: Real-time RT-PCR analyses demonstrated that the expressionsof mRNA of sXBP1and XBP1were different between controls and transgenemice at different stages. The levels of sXBP1and XBP1mRNA were moreabundant in on-set stage and end-stage transgenic mice than in age-matchedcontrols, on-set stage transgenic mice more symptomatic. Well, at every stage,the levels of ATF6mRNA did not differ between transgenic mice and controls.Conclusions: In this experiment we try to study in different stage ofhSOD1G93Atransgenic mice, the level of sXBP1and XBP1mRNA showsdiffer, it demonstrated the strength of ER stress showed difference in different stage. In on-set stage transgenic mice the sXBP1and XBP1mRNA was mostabundant, In this stage, ER stress is intense.