Changes Of ATF6 Expression In Familial Amyotrophic Lateral Sclerosis Model

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease, the motor neurons of cerebral cortex, brainstem and spinal cord anterior horn are selective loss, manifested as muscle weakness, muscle atrophy, fasciculations tremor and pyramidal tract sign. Familial amyotrophic lateral sclerosis (FALS) in 20% with Cu/Zn superoxide dismutase (SOD1) mutations, the mice with human mutant SOD1 gene have similar clinical and pathological features to ALS.Since 1991, Chain locus in chromosome 21 had been found in ALS, several other studies confirmed SOD1 gene mutation in 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 study found that some mSOD1 maintain a 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 toxic function" relevant. We 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, is the internationally recognized ALS pathogenesis and pre-clinical drug research in animal models.Recent studies have shown that ALS cerebral cortex, brainstem and spinal cord anterior horn motor neuron selective loss caused mostly by apoptosis. 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.Endoplasmic reticulum stress activating unfolded protein response (UPR), 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).UPR as occur when the endoplasmic reticulum stress caused by dissociation of protein GRP78/BIP with PREK, AFT6 and IRE-1 separation,thereby activating PREK,ATF6 and IRE-1 signaling pathwayATF6 is the typeⅡendoplasmic reticulum transmembrane protein. When endoplasmic reticulum stress, ATF6 and GRP78 dissociation and transferred to the Golgi apparatus, the Golgi protease S1P and S2P cut its transmembrane fragments produced free of 50kuN fragment (P50), P50 transfer to the nucleus and thus cause CHOP, GRP78 protein expression and ERAD, cause cell apoptosis.Recent studies show that endoplasmic reticulum stress and endoplasmic reticulum stress-induced apoptosis exist in hSOD1G93A mice spinal cord. This preliminary study found that hSOD1G93A motor cortex of the brain in mice also exists in the ATF6 endoplasmic reticulum stress signaling pathway activation.PartⅠhSOD1G93A transgenic mice breeding and identifyObjective:To obtain hSOD1G93A transgenic miceMethods:B6SJL-Tg (SOD1-G93A) 1Gur/J hemizygous males and B6SJLF1/J+/+females breeding, extracting tip of its tail to DNA, for PCR analysis identified.Results:The identified with hSOD1G93A transgenic mice and wtSOD1 mice. Conclusion:hSOD1G93A transgenic mice as an animal model of FALS study, wtSOD1 mice as an control group.PartⅡChanges of endoplasmic reticulum stress protein ATF6 in the motor cortex of hSOD1G93A transgenic miceObjective:To observe the changes of endoplasmic reticulum stress factor ATF6 in transgenic mice hSOD1G93A motor cortex, reveals endoplasmic reticulum stress in the pathogenesis of ALS.Methods:1 Experimental group:divided into four groups, wtSOD1 mice the control group 3 mice (120), pre-symptoms hSOD1G93A transgenic mice 3 mice (60 days), symptoms of hSOD1G93A transgenic mice 3 mice (100-120 days), end-stage hSOD1G93A transgenic mice 3 mice (120-130 days).2 Drawn:drawn with 10% of the chloral hydrate anesthetized mice,4% paraformaldehyde perfusion-fixed within the heart, remove the brain, with a separation of the motor cortex area, immersed in 4% paraformaldehyde, the vibration slicer Continuous slice, slice thickness 30μm.3 Immunohistochemistry:vibration slice 0.01MPBS washing three times, each 5 minutes,3%H2O2 at room temperature for 15 minutes,0.01MTBS washed three times, each 5 minutes,10% horse serum closed 1 hour, add 1:200 ATF6 polyclonal rabbit antibodies,4℃overnight incubation on shaker; adding 1:200 biotinylated goat anti-rabbit IgG at room temperature on shaker 2h; adding 1:200 horseradish HRP-streptavidin at room temperature on shaker 1 h; DAB chromogenic terminate the obvious color, stretched, conventional dehydration, coverslipping.4 Statistical analysis:experimental results to the mean + standard deviation (x±s) that the use of SAS V8.0 software for statistical analysis, analysis of variance was used to compare between the two groups and statistical results to P<0.05 for significant difference.Results:The control group, ATF6 in the cytoplasm. The number of MN which ATF6 enter into nuclei pre-symptoms compared with the control group increased significantly,symptoms,end-stage symptoms compared with the control group significantly increased, symptoms and end-stage have no significant change. Symptoms and end-stage some of the morphological appeared wrinkled.Conclusion:The early onset of 60 days existed ATF6 changes, The number of MN which ATF6 enter into nuclei increased as the disease progresses. Description of endoplasmic reticulum stress involved in the pathogenesis of ALS.