The Change Of Endoplasmic Reticulum Stress In SOD1^G93A Transgenic Mouse

Objectives：Amyotrophic lateral sclerosis （ALS） is a progressive fatalneurodegenerative disorder that primarily affects motor neurons in the cerebralcortex, brainstem and spinal cord. Pathological mechanisms such asexcitotoxicity, oxidative stress, mitochondrial dysfunction and autoimmunityhave all been implicated as causative factors in the death of the motor neurons.In recent years, many researchers have proposed that endoplasmic reticulumstress is a new idea of ALS pathogenesis. SOD1mutation is the possiblepathogenesis of familial ALS. Mutant SOD1, aggregating in the ER lumen,causes endoplasmic reticulum stress. Endoplasmic reticulum stress enhancesthe protein folding capacity of the cell to deal with mutant SOD1and decreasethe misfolded protein load of the ER. As the disease progresses, a largenumber of mutant SOD1aggregates and exceeds the capacity of theendoplasmic reticulum stress processing the misfolded proteins. Disturbanceof cell homeostasis activates endoplasmic reticulum stress-induced apoptosis,causing the death of neurons. Transgenic mice carrying a mutation （G93A） inthe superoxide dismutase1（SOD1） gene replicate most phenotypes of familialALS. It is recognized as a classic mouse model of familial ALS. In thisexperiment, we study the changes of endoplasmic reticulum stress inSOD1G^93Atransgenic mouse.Methods:1. Identification and behavior scores of transgenic miceTransgenic mice（Tg mice） expressing a mutant of human SOD1[strainB6SJL-TgN （SOD1-G93A） F1/J] mated with SOD1G^93Atransgenic mice.Their progeny were identified by polymerase chain reaction （PCR）. Observethe change of behavior in transgenic mice.2. Western blot Three groups of Tg mice at pre-symptomatic stage （60day，0point）、on-setstage （90day,2point） and end stage （120day,5point） and their age-matchedcontrol littermates were used. Each lumbar cord （L4–L5） was dissected forWestern blotting analyses and stored at-80℃before used. Extracted totalprotein, and detected the expression of ATF6αand caspase-12by western boltanalysis at different stages in SOD1G^93Atransgenic mice.3. ImmunohistochemistryThree groups of Tg mice at60day （pre-symptomatic stage）、90day （on-setstage） and120day （end stage） and their age-matched control littermates wereused. For immunohistochemical analyses, different sets of Tg and control micewere deeply anesthetized with an intraperitoneal injection of Chloral hydrate,followed by4%paraformaldehyde in0.1M phosphate buffer （pH7.4）.Transverse sections （25μm thickness） of the lumbar cords （L4–L5） wereprepared at4℃using a cryostat. Hypoglossal nerve and facial nerve nucleisections were immunostained for ATF6αand caspase-12.Results: Western blot and immunohistochemical analyses demonstratedthat the expressions of ATF6αand caspase-12were different at different stagesin control and Tg mice. The expression of ATF6αP90began to decrease aton-set stage, which had significant change at end stage in Tg mice, whilecontrol mice showed no decrease. The expression of ATF6αP50increased aton-set stage. The expression of ATF6αP50at end stage was less than that aton-set stage, and more than that at pre-symptomatic stage and in control mice.The immunohistochemical staining of ATF6αand caspase-12in hypoglossalnerve and facial nerve nuclei showed no change of immunoreactivity frompre-symptomatic stage to end stage in control mice. However, motor neuron aton-set stage began to show remarkably higher ATF6αand caspase-12immunoreactivity in their nucleus, and apoptosis occured. As for GFAP,immunoreactivity showed no change at different stages in control mice.GFAP-stained astrocytes were very few in the lumbar spinal cord of controlmice. We observed a lot of GFAP-positive staining cells in lumbar spinal cordat on-set and end stage. As the disease progressed, GFAP-stained cells gradually increased.Conclusions: A strong ER stress and ER stress-induced apoptosis occursat on-set and end stage in SOD1G^93Atransgenic mice. The selective loss ofmotor neurons affects cerebral cortex, brainstem and spinal cord. However,their lesion is different. The lumbar spinal cord has the most seriousdegeneration, followed by brainstem motor nuclei, while motor cortex isaffected very slightly, where only endoplasmic reticulum stress occurs, andalmost no significant endoplasmic reticulum stress-mediated apoptosis isdetected. We observe the proliferation of astrocytes at pre-symptomatic stagein Tg mice, and the proliferation gradually increases as the disease progresses.