Expression Of JAK2/p-JAK2 In The ALS Transgenic Mice

Objective:Amyotrophic Lateral Sclerosis（ALS）is a chronic progressive and fatal neurodegenerative disease,characterized by selective damage of motoneurons in brain and spinal cord and resulting in progressive muscle paralysis and atrophy clinically.Patients eventually died of respiratory muscle paralysis.It attacks insidiously and progresses slowly,even the exact pathogenesis is still unknown.Nowadays,there is no efficient treatment.Now,series of studies have confirmed that ALS is a neurological inflammatory disease.The inflammatory process of the nervous system is mainly caused by activated microglia,astrocytes and the complex interactions between multiple cells mediated by active substances such as cytokines,chemokines,and growth factors released by activated microglia and astrocytes.It has been found that JAK2 can bind to various inflammatory cytokines and play different roles.In addition,JAKs have been shown to be involved in the regulation of proinflammatory cytokines in many types of cells.However,there are few studies based on JAK2 in neurodegenerative diseases including ALS.This study explored the expression of JAK2 and its activated form p-JAK2 during the entire duration of ALS transgenic mice,providing a basis for the role of JAK2 in the progression of ALS disease.The mechanism will provide direction for the possible targeted treatment of ALS.Method:1 Reproduction and Identification of SOD1-G93A Transgenic MiceExperimental ALS model mice were housed in a clean environment with constant humidity,temperature,and day/night（12 h）irradiation,and were fed with growth rat food and sufficient water.The mice were crossed from B6SJL/F1 female mice and B6SJL-Tg（SOD1-G93A）1 Gur/J male mice which purchased from Jackson Laboratory（Bar Harbor,ME,USA）.DNA was extracted around 21 days after birth,and the transgenic mice with human SOD1 mutation gene was identified by gel electrophoresis and GBOX-HR automatic gel imaging system.2 Motor function score of transgenic mice4 points evaluation system for mouse motor defect score:1)Normal,no movement disorder and weight loss:4 points2)Hind leg tremor at tail suspension:3 points3)Abnormal gait:2 points4)Hind limbs on at least one side:1 point5)The mouse appears supine or lying sideways and cannot be turn over within 30 seconds:score 0Onset:two successive drops of weight and abnormal gait,scoring 2points.End stage:30 seconds on the side cannot be turned over,score 0points.3 Experimental groupsThe ALS transgenic mice were divided into six groups:30-day group,60-day group,90-day group,Onset group,End-stage group and Non-transgenic control group.（n=6 in each group）4 Tissue processing and immunohistochemical staining4.1 Tissue processingThree mice in each group were sacrificed and perfused with PBS and 4%paraformaldehyde.The lumbar enlargement was dissected and post-fixed for24 h.Then,cryoprotected in 30%sucrose and embedded in OCT compound.Twenty-five micron froze sections were cut using cryostat.4.2 Immunohistochemical stainingSlices were treated with 1%H₂O₂ for 10min and TBS for 10min,then blocked for 1h with serum followed by overnight primary antibody（JAK2,p-JAK2）incubation.After rinsing with TBST,sections were incubated in biotin secondary antibody for 1h.Rinsed again,conducted incubation in third antibody for 40min.After rinsing,use DAB reagent for visualization.Then,dehydrate the sections for 5min,and transparent for 10min.Photos were taken and the results were saved.5 Western blotEnd-stage transgenic and non-transgenic mice at the same age were sacrificed and the fresh lumbar enlargements were taken for protein extraction.BCA assay was used for protein concentration measurement.Then,after SDS-PAGE electrophoresis,membrane transference,BSA blocking,rinsing,incubation with primary antibody（JAK2,p-JAK2）and fluorescent secondary antibodies,membranes were scanned with Odyssey Imaging System.6 Statistical analysisAll data were presented as mean±SEM,all statistical analysis were conducted using SPSS 18.0 software.Independent-sample T Test was used for comparison between two groups.And P<0.05 was considered statistically significant.Results:1.The expression of JAK2 in the lumbar spinal cord of ALS transgenic mice and non-transgenic mice was not very evident,as the disease progresses,the expression of JAK2 is increasing gradually.,JAK2 expression was significantly increased in the lumbar spinal cord at end-stage transgenic mice.2.In non-transgenic mice and ALS transgenic mice before disease onset,p-JAK2 is mainly expressed in the cytoplasm of motor neurons,while expressed in the activated glial cells after disease onset.3.Compared with non-transgenic mice,expression of p-JAK2 was significantly increased in the lumbar spinal cord at end-stage transgenic mice.Conclusions:In the disease progression of ALS transgenic mice,JAK2and its activation form p-JAK2 was up-regulated,and p-JAK2 is mainly expressed in the activated glial cells after disease onset,indicating that JAK2/p-JAK2 plays an important role in the pathogenesis of ALS transgenic mice.