The Expressions Of XBP-1in Lumbar Spinal Cords Of Hsod1^G93A Transgenic Mice

Objectives:Amyotrophic lateral sclerosis（ALS） is a kind of motor neurondisease and is characterized by selective damage of motor neurons in thespinal cord ventral horn, brainstem and vertebral body.It is a progressive fatalneurodegenerative disease.Patients appear limbs weakness gradually,and showrespiratory muscle failure finally,and they usually die within3-5years.Despitesubstantial efforts, the mechanism of ALS has not been fully understood,andto clarify the mechanism is vital to devise effective therapies.Recent studiesshow that endoplasmic reticulum stres（sERS）is involved in the mechanism offamilial ALS（FALS）and sporadic ALS（SALS）.ERS happens when misfoldedproteins accumulate in the ER and the calcium cycle is disturbed.To cope withthe stress, protective reaction occurs in cells,which called unfolded proteinresponse（UPR）.There includes three sensors in the UPR, they are activatingtranscription factor6（ATF6）,inositol requiring kinase1（IRE1）anddouble-stranded RNA-activated protein kinase-like endoplasmic reticulumkinase（PERK）.Under the normal conditions,ATF6,IRE1and PERK arecombined with glucose regulated protein78（GRP78） or called bindingimmunoglobulin heavy chain protein（BIP）.When the ERS happens,the threesensors dissociate from GRP78, and then downstream pathways will beactivated to restore endoplasmic reticulum homeostasis.If the homeostasis cannot be recovered, the stress exist persistent, apoptosis will be started in cells.X-box binding protein1（XBP-1）is a important transcription factor inERS.When ERS occurs,XBP-1mRNA will be spliced by IRE1,then thespliced XBP-1mRNA translocates into the nuclear to protect the cell throughup-regulating expressions of some molecular chaperones.Studies show that,the ERS protective agent Salubrinal can alleviate the clinical manifestationand delay disease progression of mice models of ALS.The research of ERS in ALS shows a new page for studies of pathogenesis, and may provide a newpoint of view for the treatment of ALS.In the clinical,most of the ALS are sporadic,nearly5-10%arefamilial.Although sporadic forms are predominant,they have similar clinicalmanifestations,studies on FALS may provide a lot of help for mechanismillustration of SALS.Approximately20%of FALS are caused by mutantSOD1.SOD1-G93A transgenic mice are the acknowledged mice models ofFALS now.This experiment stdies the expressions of XBP-1in the lumbarspinal cords of hSOD1^G93Atransgenic mice in different periods.Methods:1Reproduction and identification of hSOD1^G93Atransgenic miceB6SJL-Tg（SOD1-G93A）1Gur/J hemizygous male mice and B6SJL F1female mice were mated,offsprings’ tails tip were cut and DNA were extractedfor PCR amplification.In the identification results,mice which had hSOD1gene were hSOD1^G93Atransgenic mice,while the others were non-transgenicmice.2Group of Experiments:hSOD1^G93Atransgenic mice were divided into sixgroups,three mice in each group,they were4weeks（28d）,5weeks（35d）,7weeks（49d）,9weeks（63d） after birth,clinical onset stage（90d） and endstage（120d）.The front four groups were presymptomatic stage.Sex andage-matched littermates were used for control groups.3To observe the expressions of XBP-1in the lumbar spinal cords ofhSOD1^G93Atransgenic mice in different periods3.1Western-blot:The lumbar spinal cords were dissected after anesthetizationand bloodletting, and were stored at-80℃.The concentration was measuredafter the total protein extraction.The total protein was separated by10%SDS-PAGE and then transferred to a PVDF membrane.The membranes wereincubated with primary antibodies at4℃overnightafter blocked by5%nonfat milk.The next day,we washed the membranes with TPBS,thenincubated them with secondary antibodies at room temperature for1h.Theexpressions of XBP-1were obtained by a membrane scanner. 3.2Immunohistochemistry:The mice were anesthetized and perfused with4%paraformaldehyde,then the lumbar spinal cords were dissected and soaked inthe4%paraformaldehyde.The fixed lumbar spinal cords were cut into25umslices by a vibratome.The slices were immersed in10%H2O2and0.3%TritonX-100in turn after washing with PBS.Then they were blocked in10%horse serum for30min at room temperature.The slices were incubated withprimary antibodies at4℃o vernight.The next day,after theslices were washedin PBS,they were incubated with secondary antibodies at room temperature for2h.Then incubated with streptavidin-horseradish enzyme labeled avidin atroom temperature for1h.At last, generally DAB stain,dehydration,neutralbalata fixation.The expressions of XBP-1in motor neurons of lumbar spinalcord anterior horn were obtained by a general microscope.3.3Immunofluorescence technique:The mice were anesthetized and perfusedwith4%paraformaldehyde,then the lumbar spinal cords were dissected andsoaked in the4%paraformaldehyde.The fixed lumbar spinal cords were cutinto25um slices by a vibratome.The slices were immersed in0.3%TritonX-100for30min at room temperature after washing with PBS. Thenthey were blocked in10%horse serum for30min at room temperature.Theslices were incubated with primary antibodies at4℃overnight.The nextday,after the slices were washed in PBS for three times （10minuteseach）,they were incubated with secondary antibodies and the nuclear dyeHoechst at room temperature for2h.At last the slices were mounted withfluorescence decay quenching agent.The expressions of XBP-1in motorneurons of lumbar spinal cord anterior horn were obtained by a laser confocalscanning microscope.4Statistical analysis:Normal test and homogeneity of variance test were doneto the results.Data obeyed to normal distribution and homogeneity of variancewas showed as mean±standard deviation （x±s）.We used SPSS17.0softwarefor statistical analysis.There had a significant difference when P<0.05.Results:1The hSOD1^G93Atransgenic mice and the non-transgenic mice were identified by PCR amplification.2The Western-blot results showed that: the expression of spliced XBP-1protein in hSOD1^G93Atransgenic mice was higher than non-transgenic mice atthe on-set and the end stage,but their had no difference between them at thepre-symptomatic stage.The amount of spliced XBP-1protein in hSOD1^G93Atransgenic mice began to increase at the on-set stage,and decreased slightly atthe end stage,but was still higher than pre-symptomatic stage groups.We didnot find difference by comparing the amount of spliced XBP-1protein inhSOD1^G93Atransgenic mice of pre-symptomatic stage and in non-transgenicmice groups of different stages.The expression of unspliced XBP-1protein inhSOD1^G93Atransgenic mice was decreased than non-transgenic mice at theon-set and the end stage, but their had no difference between them at thepre-symptomatic stage.The IHC and immunofluorescence staining results showed that: there wasa significantly higher nuclear staining in motor neurons of lumbar spinal cordanterior horn in hSOD1^G93Atransgenic mice than non-transgenic mice at theon-set and the end stage.There was no remarkable nuclear staining in both thepre-symptomatic stage hSOD1^G93Atransgenic mice and the non-transgenicmice.At the end-stage, the number of motor neurons decreased greatly inhSOD1^G93Atransgenic mice,and the cell bodies were shrunkened.Conclusions:The splicing of XBP-1occured mainly at on-set and endstage in hSOD1^G93Atransgenic mice. There had no change of XBP-1expression at the pre-symptomatic stage in hSOD1^G93Atransgenic mice. Adecrease of the number of motor neurons was found at the end-stage.ERS wasinvolved in the mechanism of hSOD1^G93Atransgenic mice.