Changes Of ATF6 Expression In Spinal Cord Of Familial Amyotrophic Lateral Sclerosis Model HSOD1^G93A Transgeneic Mouse

Objective:Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease,it characterised with the motor neurons of spinal cord anterior horn, cerebral cortex,and brainstem are selective loss.The clinicle featere is musles weekness, paralysis,and respiratory muscles affected.The Patients of ALS are dead with in 3-5years.Ninety percent to ninetyfive persent of human ALS is sporadic,and 5-10% patients are familiar ALS.Mutations in superoxide dismutase 1(SOD1) are responsible for 20% cases of familial amyotrophic lateral sclerosis (ALS).The Clinicle and Pathologicle feature of hSOD1 transgenic mice are similar to the patients of ALS.Several studies confirmed SOD1 gene mutation in farmiliar ALS, mutation is almost across the entire SOD1 molecule. Mostly missense mutations,causing expression of a protein amino acid substitution,a small number of non-missense mutations - Early termination mutation, resulting in a section of the translated sequence of lack of protein molecules.Missense mutation in exon than in the codon 4 alanine (Ala) mutation of the valine (Val) or A4V mutation is the most common mutations.SOD1 activity decreased may be the possible mechanism of ALS, resulting in free radical scavenging barrier, secondary oxidative damage motor neuron (MN).Follow-up studys found that some mSOD1 proteins maintain certain or even all of the activity.Over-expression of wild-type SOD1 (wtSOD1) does not reduce even worse symptoms of ALS transgenic mice.While the mice knockout SOD1 there are no clear symptoms of ALS, suggesting that ALS may be related to mSOD1 "get some toxic function" relevant. There has created a number of kinds of expression of human SOD1 (hSOD1) mutations in transgenic mice models, hSOD1G93A transgenic mice is the most widely used,as the internationally recognized ALS pathogenesis and pre-clinical drug research in animal models.Recent studies have shown that ERS is one of pathologis of ALS and spinal cord anterior horn,cerebral cortex,and brainstem motor neurons selective loss caused mostly by apoptosis.Apoptosis is also known as programmed cell death,refers to the body are stimulated under physiological conditions,after a variety of signal transduction leading to cell produce a series of ecological and biochemical changes,and leading to the process of cell suicide.Since 1972,John Korr first time proposed the concept of apoptosis, after thirty years of research,now known to have three major intracellular signal transduction pathways to regulate cell apoptosis: (1) mitochondrial pathway; (2) death receptor pathway; (3) endoplasmic reticulum pathway.When the unfolded protein load is more serious than the endoplasmic reticulum folding capacity,it will lead to the unfolded protein aggregating in endoplasmic reticulum cavity,and causes endoplasmic reticulum stress.The performance of the unfolded protein response (unfolded porotein response UPR),endoplasmic reticulum associated degradation (ERAD) and apoptosis. UPR include an endoplasmic reticulum molecular chaperone GRP78/BIP and three endoplasmic reticulum stress protein, the three proteins are PERK (PKR-like ER kinase),ATF6 (activating transcription factor 6) and IRE-1 (inositol-requiring enzyme 1).ATF6 is one typeⅡtransmembrane protein, its cytoplasmic part includes the bZip transcription factors and transcriptional activation region.In non-stress conditions,the region of ATF6 in the ER lumen is binding with the chaperone protein BIP,and (P90ATF6) exists by this non-active condition. After the activation, separates with BIP,and cut in Golgi body by S1P, S2P protease enzyme hydrolysis.The activated form of ATF6 (P50ATF6) can transporte to the nucleus.P50ATF6 could increases the expression of a target gene,including the BIP,CHOP/GADD153,calreticulin organizations and ERAD protein,but also increases the transcription of XBP1and the ERSE protein,ultimately lead to motor neuron apoptosis. Recent studies have shown that endoplasmic reticulum stress and endoplasmic reticulum stress induced apoptosis exist in hSOD1G93A mice spinal cord.This article in preliminary study hSOD1G93A mouse′s spinal cord wether to have in the ATF6 signal activation of endoplasmic reticulum stress, and then explore the role of endoplasmic reticulum stress in the pathogenes of ALS.Methods: The first part: hSOD1G93A transgenic mice and littermate control mice received: B6SJL-Tg (SOD1G93A) 1Gur / J male mice hemizygous and B6SJLF1 / J + / + females mated. Extracting the tip of tail to DNA,PCR analysis for identification of genes hSOD1G93A.hSOD1G93A gene identified with non-transgenic mice and hSOD1G93A transgenic mice;the positive identification mice as the experimental group,the non-transgenic mice as a negative control group.The second part: (1) three mice of experimental group hSOD1G93A asymptomatic transgenic mice (60 days),three mice of symptoms of early hSOD1G93A transgenic mice (approximately 105-115 days),three mice of end-stage hSOD1G93A transgenic mice (about 120 -130 days),three mice of non-transgenic mice as negative control group (120 days).(2)At all time points with 4% chloral hydrate anesthetized mice by intraperitoneal injection,rapid exposure of the heart, prime fixed the heart by 4% paraformaldehyde,the lumbar spinal cord removed,soaked in 4% paraformaldehyde for 48 hours,the shock slicer serial sections, each film thickness 20μm,frozen section machine serial sections,each film thickness 30μm.(3) immunofluorescence staining: frozen sections rinsed three times by 0.01M PBS,each for 5 minutes,10% H2O2 at room temperature for 15 minutes,0.01M PBS;continuous washed three times,each for 5 minutes,by 0.3% Triton-100 punch biopsy,10% horse serum closed 1 hour,add 1:200 ATF6 (SANTA CRUZ) rabbit polyclonal antibody and 1:1000 SMI32 (Sternberger Monoclonals, USA) mouse polyclonal antibody,4℃overnight incubation shaker;rinsed three times by successive 0.01M PBS, each 5 minutes,add 1:200 FITC and CY5 dark fluorescent secondary antibodies,shaking 1.5 hours at room temperature,rinsed three times by 0.01M PBS continuous,each 5 minutes, the fluorescence decay quencher were mounted,the laser confocal microscope observed.(4) Immunohistochem- ical staining:sections were 0.01M PBS continuous vibration rinsing three times, each for 5 minutes,10% H2O2 at room temperature for 15 minutes;cont- inuous rinsed three times by 0.01M PBS,each for 5 minutes,after was in 0.3% TritonX-100 punch biopsy,10% horse serum closed 1 hour,add 1:200 ATF6 (SANTA CRUZ) rabbit polyclonal antibody,4℃overnight incubation shaker; continuous rinsed three times by 0.01M PBS,each 10 minutes;add 1:200 biotinylated goat anti- rabbit IgG,at room temperature shaking 2h;0.01M PBS continuous rinsing three times,each for 5 minutes;add 1:200 streptavidin- horseradish enzyme labeled avidin at room temperature shaker 1h;DAB color- ed for 15 minutes,distilled water to terminate color,stretched routine dehydration, were mounted gelatin.Statistical analysis: The results as the mean±standard deviation ( x±s), using SPSS 17.0 software for statistical analysis, comparison between groups using non-parametric tests and analysis of T test was used to compare between the two groups, results to P <0.05 as significant difference.Results:1.The experimental animals were divided into hSOD1G93A transgenic mice and negative mice by PCR.2.Symptoms early group and end-stage group compared with control and asymptomatic group, the number of motor neurons were significantly increased which ATF6 was into the nucleus.It had statistically significant;but the number of positive cells was not significantly changed beteen early symptoms and end-stage;ATF6 expressed in motor neurons cytoplasm at the control group and asymptomatic group,the two groups was not significantly compared;and ATF6 were observed in the nucleus at the early symptoms.3.Some of the motor neurons morphology changes at the early symptoms and end-stage,shrinkage,the number of end-stage motor neurons greatly reduced.Conclusion:In the asymptomatic group (60 days) did not find endoplasmic reticulum stress factor ATF6 change.In early symptoms of ALS disease ATF6 changed significantly and increased as the disease progresses.It is indicating that endoplasmic reticulum stress partipate in the pathogenesis of ALS.However,the clear pathogenesis of ALS needs further study.