Change Of Ferritin Heavy Chain In Skeletal Muscle Of Amyotrophic Lateral Sclerosis SOD1 G93A Transgenic Mice

Objective: Amyotrophic Lateral Sclerosis (ALS) is a progressive neuron degenerative disease, involving cortical motor neurons,anterior horn cells of spinal cord , brainstem motor nuclear . Its character is the selective loss of upper and lower motor neurons, the progressive muscular dystrophy, myasthenia and pyramidal signs. Their sensory systems are not violated generally . Patients die 3-5 years after the onset of illness due to bulbar paralysis, respiratory muscle paralysis or lung infection. At present there is no effective treatment methods. Riluzole is the only approved drug for the treatment of ALS, but it can just delayed the progress of ALS, can not improve symptoms or reverse the trend of deterioration. The mechanism is not clear,but multiple mechanisms have been implicated to explain motor neuron injury including genetic and environmental factors, oxidative stress, mitochondrial dysfunction, toxicity of excitatory amino acid, imbalance of calcium homeostasis, abnormal protein aggregation. The majority of cases (90%) are sporadic (sALS), while 10% of cases are familial (fALS). The clinical expression of sporadic and familial ALS are very similar. Mutations in the gene coding for the antioxidant enzyme Cu,Zn superoxide dismutase (SOD1) are carried by 20% of fALS patients and 4% of sALS patients. Among this the G93A site is the most mutant site, and transgenic mice carrying mutant human SOD1 G93A is the most common animal model to investigate ALS in the world, because its clinical onset character is very similar with human patients .Iron is the most abundant trace element in the body,and because of its unique chemical reactivity, it plays important roles in the maintenance of celluar and organic normal metabolic process, such as delivery of oxygen, mitochondrial oxidative reaction ,the synthesis of neurotransmitter and DNA. But free iron can generate highly reactivitve hydroxy radicals through Fenton reaction , lead to oxidative stress, and damage some basic components of organism,such as lipids,proteins , DNA, and result in celluar injury. So maintanice of celluar iron balance is very important to body. There are subtle regulation mechanisms to rugulate iron in cell. Ferritin is an important protein of iron metabolism, it can combine the free iron , prevent the fenton reaction and restrict the generation of the hydroxyl radical and follow-up of oxidative stress in order to prevent cell damage. The change of ferritin represent disorder of iron metabolism in some degree. Ferritin is also an anti-oxidation protein at the downstream of Nrf2/ARE signal pathway, it can be induced and play the role of antioxidative stress.Ferritin is composed of heavy chain and light chain ,the heavy chain has the activity of ferrous oxidase, it can catalyze the soluble iron oxidized to the insoluble ferric ions, it plays important role in the rapid detoxification and the transport of iron in cells. Ferritin is regulated by the concentration of celluar iron, when the iron level is low, the expression of ferritin decrease,when the iron increases,the expression of ferritin increase.Some literature report ferric ion was detected raised in nucelus and cytoplasm in some zones of cerebrum and motor neuron in anterior horn of the spinal cords in ALS patients.Mary K found the mRNA of ferritin-H, and ferritin-L were enhanced in spinal cords at 4 months of age in SOD1 G93A transgenic mice using GeneChip. Strey CW and others found that the expression of ferritin heavy chain was increased in the homogenate of spinal cords in end stage SOD1 G93A mice compared with the control group mice using protein profiling methods, this demenstrate that the change of iron homeostasis has played an important role in the pathogenesis of ALS. Annie S. Wu found that intraperitoneal injection of FeTCPP significantly improved motor function and extended survival in SOD1 G93A mice. Similar results were seen with a second group of mice wherein treatment with FeTCPP was initiated at the onset of hindlimb weakness—roughly equivalent to the time at which treatment would begin in human patients.This experiment was used the worldly received animal model of amyotrophic lateral sclerosis—SOD1 G93A mice as study object to investigate the change of ferritin heavy chain of hindlimb skeletal muscle at different time point, look for protein marker for ALS and to provide laboratory basis for clinical diagnose and therapy .Methods: 1. the propagation of transgenic mice All animals were kept in constant temperature (25-27℃), hang wet and sterile conditions (Specific pathogen free ,SPF) environment and fed with the sterilization of SPF rodents feed particles. To maintain B6SJL-TgN (SOD1G93A) 1 Gur transgenic mice under-gene mutation, B6SJL SOD1G93A / + hemizygous males and B6SJLF1 / J females were mated , the genetic offspring mice were identified with a mutant gene determination .2. the experimental group The mice were devided into 80 days (Symptomless stage), 120 days (early symptom stage), and end stage three groups based on the morbidity law of SOD1 G93A mice. The time that SOD1 G93A mice supine 20 seconds and can not turn over was considered as end stage. Each time point was also had model group, control group 1 and control group 2. SOD1 G93A mouse was model group, Wild-type mice was control group 1, hSOD1 mice was control group 2. Each group has 3 mice at every time point.3. observe the clinical change and sacrifice the mice observe the clinical change of mice every day,and measure their weights every week. Sacrifice SOD1 G93A mice at 80 days, 120 days and end stage , Wild-type and hSOD1 mice were also sacrificed respectively in the corresponding time points.using 0.1ml 10% chloral hydrate anesthesia the mice , extract gastrocnemius of mice, put them into 1.5ml EP tubes,then to liquid nitrogen for frozing, then they were stored in refrigerator at -80℃.4.determination of ferritin heavy chain we used the western blotting method. At first extract protein of the skeletal muscle, measure the concentration ,calculate the volume that the 100μg protein needed, then carry on SDS-PAGE electrophoresis, transfer the membrane at 100 V for 2 hours, close the membrane by 5% skim milk for 1 hour , add antibody overnight at 4℃, add the anti-antibody for two hours, use Odyssey Infrared Imaging System testing the band , and calculate the ratio of ferritin heavy chain andβ-actin. The experiment data was demenstrate as mean±standard deviation ( (x|-)±s) , statistical analysis was used SAS13.0 software, statistical result is remarkable difference when P<0.05.Results: 1.Appraisal of offspring mice: PCR electrophoresis of offspring mice geneome DNA demonstrates: there is an bond located between 300-400bp(324 bp), it is PCR product of IL-2; there is an bond located between 200-300bp(236 bp), it is PCR product of SOD1 gene.2.clinical change: SOD1 G93A mice have no change compared with the control group mice at 80 days , in about 110 days limbs begin to tremor , be weak, in about 120 days SOD1 G93A mice have abnormal gait, the step chang shortly , after that one side of the hindlimb begain to be dystrophy ,then and the other side hindlimb and the hind body were involved. the hindlimb can not support body, The SOD1 G93A mice achive to end stage at about 140-150 days when they lies and not to be able to turn over for 20 seconds. Wild-type mice and hSOD1mice were very active, had no different with other normal mice .3.the level of ferritin heavy chain: compared with the Wild-type mice and hSOD1mice,the expression of ferritin heavy chain in skeletal muscle of SOD1 G93A mice did not chang at 80 days(P>0.05),but its expression increased at 120days and at end stage(P<0.050), the expression of ferritin heavy chain in skeletal muscle of Wild-type mice did not change compared with hSOD1 mice (P> 0.05).Conclusion: the SOD1 G93A mouse is the ideal animal model to investigate ALS, the expression of ferritin heavy chain in skeletal muscle of SOD1 G93A mice was increased compared with wild-type mice and hSOD1 mice at 120 days and end stage. It is speculated that up-regulation of ferritin heavy chain is a protective response of the body to the iron overload,or the result of activation of Nrf2/ARE pathway.