Changes Of P-eif2α Expression In Lumbar Spinal Of Familial Amyotrophic Lateral Sclerosis Model HSOD1^G93A Transgenic Mouse

Objectives: Amyotrophic lateral sclerosis （ALS） is the most common from ofmotor neuron disease. It is characterized by progressive degeneration of motorneurons in the primary cortex, brainstem, and spinal cord, which results inmuscle dystrophy, paralysis. The fatal event is usually failure of therespiratory muscles. At least5%to10%of all ALS cases are inherited（familial ALS） while the other cases occur sporadically. Current evidencesuggests that gene mutations have been recognized to be closely linked to thepathological mechanisms of familial ALS. Superoxide dismutase1（SOD1） isthe first familial ALS-linked gene. Approximately10%to20%of familialcases are caused by mutations in the gene encoding SOD1. There is the similarclinical and pathological mechanism between transgenic mice expressingmutant forms of human SOD1and human ALS. Therefore, many transgenicmice models carrying mutant human SOD1have been established, thehSOD1^G93Atransgenic mice which is the most widely used has beenrecognized for international animal models. Such a model would contribute toelucidating the pathological mechanism of familial ALS and establishing newtherapeutic agents. Multiple interacting factors contribute to ALS. Theseinclude protein misfolding and aggregation, excitotoxicity,neuro-inflammation, oxidative stress and mitochondrial dysfunction,cytoskeleton abnormalities and defective axonal transport besides gene mutant.Recently, endoplasmic reticulum stress （ERS） is a new pathologicalmechanism of ALS and gradually researched.Endoplasmic reticulum is an important organelle through the secretorypathway, it serves many important functions, including protein synthesis,folding and post-translational modification, the synthesis of lipids, and the regulation of calcium levels. At the same time, it exports the protein to otherorganelles. ER stress occurs when misfolded proteins accumulate in the ERlumen and ER calcium balance disorders which lead endoplasmic reticulumfolding assembly and transport proteins ability disorders. In hSOD1^G93Atransgenic mice, mutant SOD1gene expresses the mutant SOD1protein. Themutant SOD1protein is pone to aggregation in ER lumen and intracellularinclusion formation, which would lead to ERS. The mutant SOD1proteincould be cleared by unfolded protein response （UPR） and ER associateddegradation （ERAD） to alleviate the burden of ER for maintain homeostasis；however, if failed, it will lead to increased mutant SOD1aggregation, whichcause the cell death. Activation of PRK-like ER kinase （PERK）,inositol-requiring enzyme1（IRE1） and activating transcription factor6（ATF6）initiates during UPR. As the first activated signaling pathway, PERK play animportant role in pathological mechanism of ALS.In this experiment, we observed the expression of p-eif2α protein, thetarget of PERK pathway, in spinal cord of the hSOD1^G93Atransgenic mice,which proves whether PERK pathway is activated in spinal cord of thehSOD1^G93Atransgenic mice and then research the role of ERS in ALS.Methods:1. An animal model obtained and groupB6SJL-Tg（SOD1-G93A）1Gur/J male mice with B6SJLF1/J+/+femalesmated. Their progeny were identified by polymerase chain reaction （PCR） ofDNA from extracting the tip of tail to distinguish the positive mice whichexpressing a mutant of human SOD1（Tg mice） and the negative mice whichno expressing SOD1.There are six group mice in this experiment:28day group （postnatal day28）,35day group （postnatal day35）,49day group （postnatal day49）,63daygroup （postnatal day63）, pre-symptomatic group （about postnatal day90,2point）, end-stage group （about postnatal day120,5point）. The behavior of thetransgenic mice in pre-symptomatic and end-stage group assessed by standardof Snow J. There are three mice in each group and their age-matched no transgenic littermates were used.2. To observe the changes of p-eif2α in the spinal cord of hSOD1^G93Atransgenic mice2.1Western blotThe transgenic mice in each group （Tg） and their age-matched notransgenic littermates （control） were deeply anesthetized with anintraperitoneal injection of4%chloral hydrate. The lumbar spinal were newlydissected and stored at-80℃before used. Extracted total protein of eachspinal cord, we used it for western blot analysis of p-eif2α.2.2ImmunofluorescenceTg and control mice were deeply anesthetized with an intraperitonealinjection of4%chloral hydrate and then quickly fixed by the4%paraformaldehyde. The lumbar spinal of each mice were removed and soakedin4%paraformaldehyde. Serial sectioned with the frozen section machine,each slice thickness20μm. Observe the expression of p-eif2α in these slices byimmunofluorescence.2.3ImmunohistochemicalTg and control mice were deeply anesthetized with an intraperitonealinjection of4%chloral hydrate and quickly followed by4%paraformaldehydeThe spinal cord of each mice were removed and soaked in4%paraformaldehyde. Serial sectioned with shock slicer, each slice thickness25μm. The sections were immunostained for p-eif2α antibody.Results: Western blot, immunofluorescence and immunohistochemicalanalysis of lumbar spinal from mutant SOD1transgenic mice model showedthe expressions of p-eif2α. The ER stress occurs in the motor neurons duringall different stages of the transgenic SOD1G93Amice.1.The expressions ofp-eif2α at symptoms early group increased significantly, then at end stage wasless than that at symptoms early, and more than that at other stage groups andin control groups.2.Tg mice and control mice show the expression of p-eif2αin cytoplasm and process of large motor neurons in the anterior horn of spinalcord in the transgenic SOD1G93Amice. However, motor neuron at symptoms early group showed remarkable increase in p-eif2α immunoreactivity andsimilar intensity in both other Tg groups and control groups.3. It revealed asignificantly stronger staining for p-eif2α in the cytoplasm of motor neurons inanterior horn, no staining in the nucleus, especially at symptoms early group.After symptoms early stage, there were changes in the anterior horn such asorganizations appearing vacuolization, motor neurons shrinking anddecreasing in number. While no such change was observed in the other Tggroups and control groups.Conclusion: In the lumbar spinal of the transgenic SOD1G93Amice, ER stressoccur in motor neurons in anterior horn, remarkable at symptoms early andend stage. We evidence that activation of PERK pathway in spinal cord of thehSOD1^G93Atransgenic mice and then indicate that ER stress is involved in thepathogenesis of ALS.